Fusion proteins and methods thereof

ABSTRACT

The invention discloses oncogenic fusion proteins. The invention provides methods for treating gene-fusion based cancers.

This application is a continuation-in-part of International ApplicationNo. PCT/US2013/051888, filed on Jul. 24, 2013, which claims priority toU.S. Provisional Patent Application No. 61/675,006, filed on Jul. 24,2012, the content of which is hereby incorporated by reference in theirentireties. This application also claims priority to U.S. ProvisionalPatent Application No. 62/096,311, filed on Dec. 23, 2014, the contentof which is hereby incorporated by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos.R01CA101644, R01CA085628, R01CA131126, and R01CA178546 awarded by theNational Cancer Institute and R01NS061776 awarded by the NationalInstitute of Neurological Disorders and Stroke. The Government hascertain rights in the invention.

All patents, patent applications and publications cited herein arehereby incorporated by reference in their entirety. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

BACKGROUND OF THE INVENTION

Glioblastoma multiforme (GBM) is the most common form of brain cancerand among the most incurable and lethal of all human cancers. Thecurrent standard of care includes surgery, chemotherapy, and radiationtherapy. However, the prognosis of GBM remains uniformly poor. There arefew available targeted therapies and none that specifically target GBM.

The target population of GBM patients who may carry FGFR-TACC genefusions and would benefit from targeted inhibition of FGFR kinaseactivity is estimated to correspond to 6,000 patients per yearworld-wide.

SUMMARY OF THE INVENTION

The invention is based, at least in part, on the discovery of a highlyexpressed class of gene fusions in GBM, which join the tyrosine kinasedomain of FGFR genes to the TACC domain of TACC1 or TACC3. The inventionis based, at least in part, on the finding that FGFR-TACC fusionsidentify a subset of GBM patients who will benefit from targetedinhibition of the tyrosine kinase activity of FGFR. Identification offusions of FGFR and TACC genes in glioblastoma patients and othersubjects afflicted with a gene-fusion associated cancer (such as anepithelial cancer) are useful therapeutic targets.

The invention is also based, at least in part, on the discovery of genefusions joining the tyrosine kinase domain of FGFR genes to the TACCdomain of TACC1 or TACC3 in grade II and III glioma, The invention isbased, at least in part, on the finding that FGFR-TACC fusions identifya subset of grade II and III glioma patients who will benefit fromtargeted inhibition of the tyrosine kinase activity of FGFR.Identification of fusions of FGFR and TACC genes in glioma patients areuseful therapeutic targets.

An aspect of the invention provides for a purified fusion proteincomprising a tyrosine kinase domain of an FGFR protein fused to apolypeptide that constitutively activates the tyrosine kinase domain ofthe FGFR protein. In one embodiment, the FGFR protein is FGFR1, FGFR2,FGFR3, or FGR4. In another embodiment, the purified fusion protein isessentially free of other human proteins.

An aspect of the invention provides for a purified fusion proteincomprising a transforming acidic coiled-coil (TACC) domain fused to apolypeptide with a tyrosine kinase domain, wherein the TACC domainconstitutively activates the tyrosine kinase domain. In one embodiment,the TACC protein is TACC1, TACC2, or TACC3. In another embodiment, thepurified fusion protein is essentially free of other human proteins.

An aspect of the invention provides for a purified fusion proteincomprising the tyrosine kinase domain of an FGFR protein fused 5′ to theTACC domain of a transforming acidic coiled-coil-containing (TACC)protein. In one embodiment, the FGFR protein is FGFR1, FGFR2, FGFR3, orFGR4. In another embodiment, the TACC protein is TACC1, TACC2, or TACC3.In another embodiment, the purified fusion protein is essentially freeof other human proteins.

An aspect of the invention provides for a purified fusion proteinencoded by an FGFR1-TACC1 nucleic acid, wherein FGFR1-TACC1 comprises acombination of exons 1-17 of FGFR1 located on human chromosome 8p11spliced 5′ to a combination of exons 7-13 of TACC1 located on humanchromosome 8p11, wherein a genomic breakpoint occurs in any one of exons1-17 of FGFR1 and any one of exons 7-13 of TACC1. In another embodiment,the purified fusion protein is essentially free of other human proteins.

An aspect of the invention provides for a purified fusion proteinencoded by an FGFR2-TACC2 nucleic acid, wherein FGFR2-TACC2 comprises acombination of any exons 1-18 of FGFR2 located on human chromosome 10q26spliced 5′ to a combination of any exons 1-23 of TACC2 located on humanchromosome 10q26. In another embodiment, the purified fusion protein isessentially free of other human proteins.

An aspect of the invention provides for a purified fusion proteinencoded by an FGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3 comprises acombination of exons 1-16 of FGFR3 located on human chromosome 4p16spliced 5′ to a combination of exons 8-16 of TACC3 located on humanchromosome 4p16, wherein a genomic breakpoint occurs in any one of exons1-16 of FGFR3 and any one of exons 8-16 of TACC3. In another embodiment,the purified fusion protein is essentially free of other human proteins.

An aspect of the invention provides for a purified fusion proteinencoded by an FGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3 comprises acombination of exons 1-18 of FGFR3 located on human chromosome 4p16spliced 5′ to a combination of exons 4-16 of TACC3 located on humanchromosome 4p16, wherein a genomic breakpoint occurs in any one of exons1-18 of FGFR3 and any one of exons 4-16 of TACC3. In another embodiment,the purified fusion protein is essentially free of other human proteins.

An aspect of the invention provides for a purified fusion proteinencoded by an FGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3 comprises acombination of exons 1-16 of FGFR3 located on human chromosome 4p16spliced 5′ to a combination of exons 8-16 of TACC3 located on humanchromosome 4p16, wherein a genomic breakpoint occurs in any one ofintrons 1-16 of FGFR3 and any one of exons 8-16 of TACC3. In anotherembodiment, the purified fusion protein is essentially free of otherhuman proteins.

An aspect of the invention provides for a purified fusion proteinencoded by an FGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3 comprises acombination of exons 1-18 of FGFR3 located on human chromosome 4p16spliced 5′ to a combination of exons 4-16 of TACC3 located on humanchromosome 4p16, wherein a genomic breakpoint occurs in any one ofintrons 1-18 of FGFR3 and any one of exons 4-16 of TACC3. In anotherembodiment, the purified fusion protein is essentially free of otherhuman proteins.

An aspect of the invention provides for a purified fusion proteinencoded by an FGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3 comprises acombination of exons 1-16 of FGFR3 located on human chromosome 4p16spliced 5′ to a combination of exons 8-16 of TACC3 located on humanchromosome 4p16, wherein a genomic breakpoint occurs in any one of exons1-16 of FGFR3 and any one of introns 7-16 of TACC3. In anotherembodiment, the purified fusion protein is essentially free of otherhuman proteins.

An aspect of the invention provides for a purified fusion proteinencoded by an FGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3 comprises acombination of exons 1-18 of FGFR3 located on human chromosome 4p16spliced 5′ to a combination of exons 4-16 of TACC3 located on humanchromosome 4p16, wherein a genomic breakpoint occurs in any one of exons1-18 of FGFR3 and any one of introns 3-16 of TACC3. In anotherembodiment, the purified fusion protein is essentially free of otherhuman proteins.

An aspect of the invention provides for a purified fusion proteinencoded by an FGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3 comprises acombination of exons 1-16 of FGFR3 located on human chromosome 4p16spliced 5′ to a combination of exons 8-16 of TACC3 located on humanchromosome 4p16, wherein a genomic breakpoint occurs in any one ofintrons 1-16 of FGFR3 and any one of introns 7-16 of TACC3. In anotherembodiment, the purified fusion protein is essentially free of otherhuman proteins.

An aspect of the invention provides for a purified fusion proteinencoded by an FGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3 comprises acombination of exons 1-18 of FGFR3 located on human chromosome 4p16spliced 5′ to a combination of exons 4-16 of TACC3 located on humanchromosome 4p16, wherein a genomic breakpoint occurs in any one ofintrons 1-18 of FGFR3 and any one of introns 3-16 of TACC3. In anotherembodiment, the purified fusion protein is essentially free of otherhuman proteins.

An aspect of the invention provides for a synthetic nucleic acidencoding the fusion proteins described above.

An aspect of the invention provides for a purified FGFR3-TACC3 fusionprotein comprising SEQ ID NO: 79, 158, 159, 160, 161, 539, 540, 541,542, 543, 544, 545, 546, 547. In another embodiment, the purified fusionprotein is essentially free of other human proteins.

An aspect of the invention provides for a purified FGFR3-TACC3 fusionprotein having a genomic breakpoint comprising at least 3 consecutiveamino acids from amino acids 730-758 of SEQ ID NO: 90 and comprising atleast 3 consecutive amino acids from amino acids 549-838 of SEQ ID NO:92. In another embodiment, the purified fusion protein is essentiallyfree of other human proteins.

An aspect of the invention provides for a purified FGFR3-TACC3 fusionprotein having a genomic breakpoint comprising at least 3 consecutiveamino acids from amino acids 730-781 of SEQ ID NO: 90 and comprising atleast 3 consecutive amino acids from amino acids 432-838 of SEQ ID NO:92. In another embodiment, the purified fusion protein is essentiallyfree of other human proteins.

An aspect of the invention provides for a purified FGFR3-TACC3 fusionprotein having a genomic breakpoint comprising SEQ ID NO: 78. In anotherembodiment, the purified fusion protein is essentially free of otherhuman proteins.

An aspect of the invention provides for a purified FGFR3-TACC3 fusionprotein having a genomic breakpoint comprising any one of SEQ ID NOS:85, 86, 87, 89, 516 or 518. In another embodiment, the purified fusionprotein is essentially free of other human proteins.

An aspect of the invention provides for a purified FGFR1-TACC1 fusionprotein comprising SEQ ID NO: 150. In another embodiment, the purifiedfusion protein is essentially free of other human proteins.

An aspect of the invention provides for a purified FGFR1-TACC1 fusionprotein having a genomic breakpoint comprising at least 3 consecutiveamino acids from amino acids 746-762 of SEQ ID NO: 146 and comprising atleast 3 consecutive amino acids from amino acids 572-590 of SEQ ID NO:148. In another embodiment, the purified fusion protein is essentiallyfree of other human proteins.

An aspect of the invention provides for a purified FGFR1-TACC1 fusionprotein having a genomic breakpoint comprising at least 3 consecutiveamino acids from amino acids 746-762 of SEQ ID NO: 146 and comprising atleast 3 consecutive amino acids from amino acids 571-590 of SEQ ID NO:148. In another embodiment, the purified fusion protein is essentiallyfree of other human proteins.

An aspect of the invention provides for a purified FGFR1-TACC1 fusionprotein having a genomic breakpoint comprising SEQ ID NO: 88. In anotherembodiment, the purified fusion protein is essentially free of otherhuman proteins.

An aspect of the invention provides for a purified DNA encoding anFGFR3-TACC3 fusion protein comprising SEQ ID NO: 94, 530, 531, 532, 533,534, 535, 536, 537, or 538. In another embodiment, the purified fusionprotein is essentially free of other human proteins. An aspect of theinvention provides for a purified cDNA encoding an FGFR3-TACC3 fusionprotein comprising SEQ ID NO: 94, 530, 531, 532, 533, 534, 535, 536,537, or 538.

An aspect of the invention provides for a synthetic nucleic acidencoding an FGFR3-TACC3 fusion protein having a genomic breakpointcomprising at least 9 consecutive in-frame nucleotides from nucleotides2443-2530 of SEQ ID NO: 91 and comprising at least 9 consecutivein-frame nucleotides from nucleotides 1800-2847 of SEQ ID NO: 93.

An aspect of the invention provides for a synthetic nucleic acidencoding an FGFR3-TACC3 fusion protein having a genomic breakpointcomprising any one of SEQ ID NOS: 1-77, or 519-527.

An aspect of the invention provides for a synthetic nucleic acidencoding an FGFR1-TACC1 fusion protein comprising SEQ ID NO: 151.

An aspect of the invention provides for a synthetic nucleic acidencoding an FGFR1-TACC1 fusion protein having a genomic breakpointcomprising at least 9 consecutive in-frame nucleotides from nucleotides3178-3228 of SEQ ID NO: 147 and comprising at least 9 consecutivein-frame nucleotides from nucleotides 2092-2794 of SEQ ID NO: 149.

An aspect of the invention provides for a synthetic nucleic acidencoding an FGFR1-TACC1 fusion protein having a genomic breakpointcomprising SEQ ID NO: 83.

An aspect of the invention provides for an antibody or antigen-bindingfragment thereof, that specifically binds to a purified fusion proteincomprising a tyrosine kinase domain of an FGFR protein fused to apolypeptide that constitutively activates the tyrosine kinase domain ofthe FGFR protein. In one embodiment, the FGFR protein is FGFR1, FGFR2,FGFR3, or FGFR4. In another embodiment, the fusion protein is anFGFR-TACC fusion protein. In a further embodiment, the FGFR-TACC fusionprotein is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3. In someembodiments, the FGFR1-TACC1 fusion protein comprises the amino acidsequence of SEQ ID NO: 150. In other embodiments, the FGFR3-TACC3 fusionprotein comprises the amino acid sequence of SEQ ID NO: 79, 158, 159,160, 161, 539, 540, 541, 542, 543, 544, 545, 546, or 547.

An aspect of the invention provides for a composition for decreasing ina subject the expression level or activity of a fusion proteincomprising the tyrosine kinase domain of an FGFR protein fused to apolypeptide that constitutively activates the tyrosine kinase domain ofthe FGFR protein, the composition in an admixture of a pharmaceuticallyacceptable carrier comprising an inhibitor of the fusion protein. In oneembodiment, the fusion protein is an FGFR-TACC fusion protein. Inanother embodiment, the inhibitor comprises an antibody thatspecifically binds to a FGFR-TACC fusion protein or a fragment thereof;a small molecule that specifically binds to a FGFR protein; a smallmolecule that specifically binds to a TACC protein; an antisense RNA orantisense DNA that decreases expression of a FGFR-TACC fusionpolypeptide; a siRNA that specifically targets a FGFR-TACC fusion gene;or a combination of the listed inhibitors. In a further embodiment, theFGFR protein is FGFR1, FGFR2, FGFR3, or FGFR4. In some embodiments, theFGFR-TACC fusion protein is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3. Inother embodiments, the small molecule that specifically binds to a FGFRprotein comprises AZD4547, NVP-BGJ398, PD173074, NF449, TK1258,BIBF-1120, BMS-582664, AZD-2171, TSU68, AB1010, AP24534, E-7080,LY2874455, or a combination of the listed small molecules. In otherembodiments, the small molecule that specifically binds to a FGFRprotein comprises an oral pan-FGFR tyrosine kinase inhibitor. In otherembodiments, the small molecule that specifically binds to a FGFRprotein comprises JNJ-42756493.

An aspect of the invention provides for a method for decreasing in asubject in need thereof the expression level or activity of a fusionprotein comprising the tyrosine kinase domain of an FGFR protein fusedto a polypeptide that constitutively activates the tyrosine kinasedomain of the FGFR protein. In one embodiment, the method comprisesadministering to the subject a therapeutic amount of a composition fordecreasing the expression level or activity in a subject of a fusionprotein comprising the tyrosine kinase domain of an FGFR protein fusedto a polypeptide that constitutively activates the tyrosine kinasedomain of the FGFR protein. In one embodiment, the method comprisesobtaining a sample from the subject to determine the level of expressionof an FGFR fusion molecule in the subject. In some embodiments, thesample is incubated with an agent that binds to an FGFR fusion molecule,such as an antibody, a probe, a nucleic acid primer, and the like. Inone embodiment, the detection or determining comprises nucleic acidsequencing, selective hybridization, selective amplification, geneexpression analysis, or a combination thereof. In another embodiment,the detection or determination comprises protein expression analysis,for example by western blot analysis, ELISA, immunostaining, or otherantibody detection methods. In a further embodiment, the methodcomprises determining whether the fusion protein expression level oractivity is decreased compared to fusion protein expression level oractivity prior to administration of the composition, thereby decreasingthe expression level or activity of the fusion protein. In oneembodiment, the fusion protein is an FGFR-TACC fusion protein. In afurther embodiment, the FGFR protein is FGFR1, FGFR2, FGFR3, or FGFR4.In some embodiments, the FGFR-TACC fusion protein is FGFR1-TACC1,FGFR2-TACC2, or FGFR3-TACC3. In one embodiment, the composition fordecreasing the expression level or activity of a fusion proteincomprises an antibody that specifically binds to a FGFR-TACC fusionprotein or a fragment thereof; a small molecule that specifically bindsto a FGFR protein; a small molecule that specifically binds to a TACCprotein; an antisense RNA or antisense DNA that decreases expression ofa FGFR-TACC fusion polypeptide; a siRNA that specifically targets aFGFR-TACC fusion gene; or a combination of the listed inhibitors. In afurther embodiment, the FGFR protein is FGFR1, FGFR2, FGFR3, or FGFR4.In some embodiments, the FGFR-TACC fusion protein is FGFR1-TACC1,FGFR2-TACC2, or FGFR3-TACC3. In other embodiments, the small moleculethat specifically binds to a FGFR protein comprises AZD4547, NVP-BGJ398,PD173074, NF449, TK1258, BIBF-1120, BMS-582664, AZD-2171, TSU68, AB1010,AP24534, E-7080, LY2874455, or a combination of the small moleculeslisted. In other embodiments, the small molecule that specifically bindsto a FGFR protein comprises an oral pan-FGFR tyrosine kinase inhibitor.In other embodiments, the small molecule that specifically binds to aFGFR protein comprises JNJ-42756493.

An aspect of the invention provides for a method for treating agene-fusion associated cancer in a subject in need thereof, the methodcomprising administering to the subject an effective amount of a FGFRfusion molecule inhibitor. In one embodiment, the gene-fusion associatedcancer comprises an epithelial cancer. In one embodiment, thegene-fusion associated cancer comprises glioblastoma multiforme, breastcancer, lung cancer, prostate cancer, or colorectal carcinoma. In oneembodiment, the gene-fusion associated cancer comprises bladdercarcinoma, squamous lung carcinoma and head and neck carcinoma. In oneembodiment, the gene-fusion associated cancer comprises glioma. In oneembodiment, the gene-fusion associated cancer comprises grade II or IIIglioma. In one embodiment, the gene-fusion associated cancer comprisesIDH wild-type grade II or III glioma. In one embodiment, the methodcomprises obtaining a sample from the subject to determine the level ofexpression of an FGFR fusion molecule in the subject. In someembodiments the sample from the subject is a tissue sample. In someembodiments, the sample is a paraffin embedded tissue section. In someembodiments, the tissue sample from the subject is a tumor sample. Insome embodiments, the sample is incubated with an agent that binds to anFGFR fusion molecule, such as an antibody, a probe, a nucleic acidprimer, and the like. In one embodiment, the detection or determiningcomprises nucleic acid sequencing, selective hybridization, selectiveamplification, gene expression analysis, or a combination thereof. Inanother embodiment, the detection or determination comprises proteinexpression analysis, for example by western blot analysis, ELISA,immunostaining, or other antibody detection methods. In anotherembodiment, the FGFR fusion protein comprises an FGFR protein fused to apolypeptide that constitutively activates the tyrosine kinase domain ofthe FGFR protein. In one embodiment, the fusion protein is an FGFR-TACCfusion protein. In another embodiment, the inhibitor comprises anantibody that specifically binds to a FGFR-TACC fusion protein or afragment thereof; a small molecule that specifically binds to a FGFRprotein; a small molecule that specifically binds to a TACC protein; anantisense RNA or antisense DNA that decreases expression of a FGFR-TACCfusion polypeptide; a siRNA that specifically targets a FGFR-TACC fusiongene; or a combination of the listed inhibitors. In a furtherembodiment, the FGFR protein is FGFR1, FGFR2, FGFR3, or FGFR4. In someembodiments, the FGFR-TACC fusion protein is FGFR1-TACC1, FGFR2-TACC2,or FGFR3-TACC3. In other embodiments, the small molecule thatspecifically binds to a FGFR protein comprises AZD4547, NVP-BGJ398,PD173074, NF449, TK1258, BIBF-1120, BMS-582664, AZD-2171, TSU68, AB1010,AP24534, E-7080, LY2874455, or a combination of the small moleculeslisted. In other embodiments, the small molecule that specifically bindsto a FGFR protein comprises an oral pan-FGFR tyrosine kinase inhibitor.In other embodiments, the small molecule that specifically binds to aFGFR protein comprises JNJ-42756493.

An aspect of the invention provides for a method of decreasing growth ofa solid tumor in a subject in need thereof, the method comprisingadministering to the subject an effective amount of a FGFR fusionmolecule inhibitor, wherein the inhibitor decreases the size of thesolid tumor. In one embodiment, the solid tumor comprises glioblastomamultiforme, breast cancer, lung cancer, prostate cancer, or colorectalcarcinoma. In one embodiment, the solid tumor comprises bladdercarcinoma, squamous lung carcinoma and head and neck carcinoma. In oneembodiment, the solid tumor comprises glioma. In one embodiment, thesolid tumor comprises grade II or III glioma. In one embodiment, thesolid tumor comprises IDH wild-type grade II or III glioma. In oneembodiment, the method comprises obtaining a sample from the subject todetermine the level of expression of an FGFR fusion molecule in thesubject. In some embodiments the sample from the subject is a tissuesample. In some embodiments, the sample is a paraffin embedded tissuesection. In some embodiments, the tissue sample from the subject is atumor sample. In some embodiments, the sample is incubated with an agentthat binds to an FGFR fusion molecule, such as an antibody, a probe, anucleic acid primer, and the like. In one embodiment, the detection ordetermining comprises nucleic acid sequencing, selective hybridization,selective amplification, gene expression analysis, or a combinationthereof. In another embodiment, the detection or determination comprisesprotein expression analysis, for example by western blot analysis,ELISA, immunostaining, or other antibody detection methods. In anotherembodiment, the FGFR fusion protein comprises an FGFR protein fused to apolypeptide that constitutively activates the tyrosine kinase domain ofthe FGFR protein. In one embodiment, the fusion protein is an FGFR-TACCfusion protein. In another embodiment, the inhibitor comprises anantibody that specifically binds to a FGFR-TACC fusion protein or afragment thereof; a small molecule that specifically binds to a FGFRprotein; a small molecule that specifically binds to a TACC protein; anantisense RNA or antisense DNA that decreases expression of a FGFR-TACCfusion polypeptide; a siRNA that specifically targets a FGFR-TACC fusiongene; or a combination of the listed inhibitors. In a furtherembodiment, the FGFR protein is FGFR1, FGFR2, FGFR3, or FGFR4. In someembodiments, the FGFR-TACC fusion protein is FGFR1-TACC1, FGFR2-TACC2,or FGFR3-TACC3. In other embodiments, the small molecule thatspecifically binds to a FGFR protein comprises AZD4547, NVP-BGJ398,PD173074, NF449, TK1258, BIBF-1120, BMS-582664, AZD-2171, TSU68, AB1010,AP24534, E-7080, LY2874455, or a combination of the small moleculeslisted. In other embodiments, the small molecule that specifically bindsto a FGFR protein comprises an oral pan-FGFR tyrosine kinase inhibitor.In other embodiments, the small molecule that specifically binds to aFGFR protein comprises JNJ-42756493.

An aspect of the invention provides for a diagnostic kit for determiningwhether a sample from a subject exhibits a presence of a FGFR fusion,the kit comprising at least one oligonucleotide that specificallyhybridizes to a FGFR fusion, or a portion thereof. In one embodiment,the oligonucleotides comprise a set of nucleic acid primers or in situhybridization probes. In another embodiment, the oligonucleotidecomprises SEQ ID NO: 162, 163, 164, 165, 166, 167, 168, 169, 495, 496,497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510 ora combination of the listed oligonucleotides. In one embodiment, theprimers prime a polymerase reaction only when a FGFR fusion is present.In another embodiment, the determining comprises gene sequencing,selective hybridization, selective amplification, gene expressionanalysis, or a combination thereof. In a further embodiment, theFGFR-fusion is an FGFR-TACC fusion. In some embodiments, the FGFR isFGFR1, FGFR2, FGFR3, or FGFR4. In other embodiments, the FGFR-TACCfusion is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3.

An aspect of the invention provides for a diagnostic kit for determiningwhether a sample from a subject exhibits a presence of a FGFR fusionprotein, the kit comprising an antibody that specifically binds to aFGFR fusion protein comprising SEQ ID NO: 79, 85, 86, 87, 88, 89, 150,158, 159, 160, 161, 516, 518, 539, 540, 541, 542, 543, 544, 545, 546, or547 wherein the antibody will recognize the protein only when a FGFRfusion protein is present. In one embodiment, the antibody directed toand FGFR fusion comprising SEQ ID NO: 79, 85, 86, 87, 88, 89, 150, 158,159, 160, 161, 516, 518 539, 540, 541, 542, 543, 544, 545, 546, or 547.In a further embodiment, the FGFR-fusion is an FGFR-TACC fusion. In someembodiments, the FGFR is FGFR1, FGFR2, FGFR3, or FGFR4. In otherembodiments, the FGFR-TACC fusion is FGFR1-TACC1, FGFR2-TACC2, orFGFR3-TACC3. In some embodiments the sample from the subject is a tissuesample. In some embodiments, the sample is a paraffin embedded tissuesection. In some embodiments, the tissue sample from the subject is atumor sample.

An aspect of the invention provides for a method for detecting thepresence of a FGFR fusion in a human subject. In one embodiment, themethod comprises obtaining a biological sample from the human subject.In some embodiments the sample from the subject is a tissue sample. Insome embodiments, the sample is a paraffin embedded tissue section. Insome embodiments, the tissue sample from the subject is a tumor sample.In some embodiments, the sample is incubated with an agent that binds toan FGFR fusion molecule, such as an antibody. In another embodiment, thedetection or determination comprises protein expression analysis, forexample by western blot analysis, ELISA, immunostaining or otherantibody detection methods. In some embodiments, the method furthercomprises assessing whether to administer a FGFR fusion moleculeinhibitor based on the expression pattern of the subject. In furtherembodiments, the method comprises administering a FGFR fusion moleculeinhibitor to the subject. In other embodiments, the FGFR fusion moleculeinhibitor comprises an oral pan-FGFR tyrosine kinase inhibitor. In otherembodiments, the FGFR fusion molecule inhibitor comprises JNJ-42756493.In another embodiment, the method comprises detecting whether or notthere is a FGFR fusion present in the subject. In one embodiment, thedetecting comprises measuring FGFR fusion protein levels by ELISA usingan antibody directed to SEQ ID NO: 79, 85, 86, 87, 88, 89, 150, 158,159, 160, 161, 516, 518 539, 540, 541, 542, 543, 544, 545, 546, or 547;western blot using an antibody directed to SEQ ID NO: 79, 85, 86, 87,88, 89, 150, 158, 159, 160, 161, 516, 518 539, 540, 541, 542, 543, 544,545, 546, or 547; immunostaining using an antibody directed to SEQ IDNO: 79, 85, 86, 87, 88, 89, 150, 158, 159, 160, 161, 516, 518, 539, 540,541, 542, 543, 544, 545, 546, or 547; mass spectroscopy, isoelectricfocusing, or a combination of the listed methods. In some embodiments,the FGFR-fusion is an FGFR-TACC fusion. In other embodiments, the FGFRis FGFR1, FGFR2, FGFR3, or FGFR4. In other embodiments, the FGFR-TACCfusion is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3.

An aspect of the invention provides for a method for detecting thepresence of a FGFR fusion in a human subject. In one embodiment, themethod comprises obtaining a biological sample from a human subject. Insome embodiments, the sample is incubated with an agent that binds to anFGFR fusion molecule, such as a probe, a nucleic acid primer, and thelike. In other embodiments, the detection or determination comprisesnucleic acid sequencing, selective hybridization, selectiveamplification, gene expression analysis, or a combination thereof. Insome embodiments, the method further comprises assessing whether toadminister a FGFR fusion molecule inhibitor based on the expressionpattern of the subject. In further embodiments, the method comprisesadministering a FGFR fusion molecule inhibitor to the subject. Inanother embodiment, the method comprises detecting whether or not thereis a nucleic acid sequence encoding a FGFR fusion protein in thesubject. In one embodiment, the nucleic acid sequence comprises any oneof SEQ ID NOS: 1-77, 80-84, 95-145, 515, 517, 519-527, or 530-538. Inanother embodiment, the detecting comprises using hybridization,amplification, or sequencing techniques to detect a FGFR fusion. In afurther embodiment, the amplification uses primers comprising SEQ ID NO:162, 163, 164, 165, 166, 167, 168, 169, 495, 496, 497, 498, 499, 500,501, 502, 503, 504, 505, 506, 507, 508, 509 or 510. In some embodiments,the FGFR-fusion is an FGFR-TACC fusion. In other embodiments, the FGFRis FGFR1, FGFR2, FGFR3, or FGFR4. In other embodiments, the FGFR-TACCfusion is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3.

An aspect of the invention provides for a method of initiating oncogenictransformation in vitro. The method comprises (a) transducing cellscultured in vitro with FGFR-TACC fusion DNA; and (b) determining whetherthe cells acquire the ability to grow in anchorage-independentconditions, form multi-layered foci, or a combination thereof.

An aspect of the invention provides for a method of initiating oncogenictransformation in vivo. The method comprises (a) transducing cellscultured in vitro with FGFR-TACC fusion DNA; (b) injecting a mouse withthe transduced cells; and (c) determining whether a tumor grows in themouse. In one embodiment, the injecting is a subcutaneous orintracranial injection.

An aspect of the invention provides a method of identifying a compoundthat decreases the oncogenic activity of a FGFR-TACC fusion. The methodcomprises (a) transducing a cell cultured in vitro with FGFR-TACC DNA;(b) contacting a cell with a ligand source for an effective period oftime; and (c) determining whether the cells acquire the ability to growin anchorage-independent conditions, form multi-layered foci, or acombination thereof, compared to cells cultured in the absence of thetest compound.

In one embodiment, the method can comprise contacting a sample from thesubject with an antibody specific for a FGFR fusion molecule, anddetermining the presence of an immune complex. In another embodiment,the method can comprise contacting a sample from the subject with anantibody specific for a FGFR molecule, or a TACC molecule, anddetermining the presence of an immune complex. In another embodiment,the antibody can recognize the FGFR3 C-terminal region, or the TACC3N-terminal region, or a combination thereof. In another embodiment, theantibody can recognize the FGFR3 C-terminal region, or the TACC3N-terminal region, or a combination thereof. In another embodiment, themethod can comprise contacting a sample from the subject with anantibody specific for a FGFR molecule, or a TACC molecule, or a FGFRfusion molecule, and determining the amount of an immune complex formedcompared to the amount of immune complex formed in non-tumor cells ortissue, wherein an increased amount of an immune complex indicates thepresence of an FGFR fusion.

In one embodiment, the method can comprise contacting a sample from thesubject with primers specific for a FGFR fusion molecule, anddetermining the presence of an PCR product. In another embodiment, themethod can comprise contacting a sample from the subject with primerspecific for a FGFR molecule, or a TACC molecule, and determining thepresence of a PCR product. In another embodiment, the primers canrecognize the nucleic acids encoding a FGFR3 C-terminal region, ornucleic acids encoding a TACC3 N-terminal region, or a combinationthereof. In another embodiment, the method can comprise contacting asample from the subject with primers specific for a FGFR molecule, or aTACC molecule, or a FGFR fusion molecule, and determining the amount ofPCR product formed compared to the amount of PCR product formed innon-tumor cells or tissue, wherein an increased amount of PCR productindicates the presence of an FGFR fusion.

An aspect of the invention provides for a purified fusion proteincomprising the tyrosine kinase domain of an FGFR protein fused to theTACC domain of a transforming acidic coiled-coil-containing (TACC)protein. In one embodiment, the FGFR protein is FGFR1, FGFR2, FGFR3, orFGFR4. In one embodiment, the TACC protein is TACC1, TACC2, or TACC3. Inone embodiment, the fusion protein is FGFR1-TACC1, FGFR2-TACC2, orFGFR3-TACC3. In one embodiment, the fusion protein comprises SEQ ID NO:79, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQID NO: 539, SEQ ID NO: 540, SEQ ID NO: 541, SEQ ID NO: 542, SEQ ID NO:543, SEQ ID NO: 545, SEQ ID NO: 546, or SEQ ID NO: 547. In oneembodiment, the fusion protein has a breakpoint comprising at least 3consecutive amino acids from amino acids 730-758 of SEQ ID NO: 90 andcomprising at least 3 consecutive amino acids from amino acids 549-838of SEQ ID NO: 92. In one embodiment, the fusion protein has a breakpointcomprising SEQ ID NO: 78, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87,SEQ ID NO:89, SEQ ID NO: 516, or SEQ ID NO:518. In one embodiment, thefusion protein comprises SEQ ID NO: 150. In one embodiment, the fusionprotein has a breakpoint comprising at least 3 consecutive amino acidsfrom amino acids 746-762 of SEQ ID NO: 146 and comprising at least 3consecutive amino acids from amino acids 572-590 of SEQ ID NO: 148. Inone embodiment, the fusion protein has a breakpoint comprising SEQ IDNO: 88.

An aspect of the invention provides for a cDNA encoding a fusion proteincomprising the tyrosine kinase domain of FGFR fused to the TACC domainof TACC. In one embodiment the FGFR is FGFR1, FGFR2, FGFR3, or FGFR4. Inone embodiment, the TACC is TACC1, TACC2, or TACC3. In one embodiment,the fusion protein is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3. In oneembodiment, the cDNA comprises SEQ ID NO: 94, SEQ ID NO: 530, SEQ ID NO:531, SEQ ID NO: 532, SEQ ID NO: 533, SEQ ID NO: 534, SEQ ID NO: 535, SEQID NO: 536, SEQ ID NO: 537 or SEQ ID NO: 538. In one embodiment, thecDNA has a breakpoint comprising at least 9 consecutive in-framenucleotides from nucleotides 2443-2530 of SEQ ID NO: 91 and comprisingat least 9 consecutive in-frame nucleotides from nucleotides 1800-2847of SEQ ID NO: 93. In one embodiment, the cDNA has a breakpointcomprising any one of SEQ ID NOs: 1-77, or SEQ ID NOs: 519-527. In oneembodiment, the cDNA comprises SEQ ID NO: 151. In one embodiment, thecDNA has a breakpoint comprising at least 9 consecutive in-framenucleotides from nucleotides 3178-3228 of SEQ ID NO: 147 and comprisingat least 9 consecutive in-frame nucleotides from nucleotides 2092-2794of SEQ ID NO: 149. In one embodiment, the cDNA has a breakpointcomprising SEQ ID NO: 83. In one embodiment, the cDNA comprises acombination of exons 1-16 of FGFR3 spliced 5′ to a combination of exons8-16 of TACC3, wherein a breakpoint occurs in: a) any one of exons 1-16of FGFR3 and any one of exons 8-16 of TACC3; b) any one of introns 1-16of FGFR3 and any one of exons 8-16 of TACC3; c) any one of exons 1-16 ofFGFR3 and any one of introns 7-16 of TACC3; or d) any one of introns1-16 of FGFR3 and any one of introns 7-16 of TACC3. In one embodiment,the cDNA comprises a combination of exons 1-17 of FGFR1 spliced 5′ to acombination of exons 7-13 of TACC1, wherein a breakpoint occurs in anyone of exons 1-17 of FGFR3 and any one of exons 7-13 of TACC3. In oneembodiment, the cDNA comprises a combination of exons 1-18 of FGFR2spliced 5′ to a combination of exons 1-23 of TACC2.

BRIEF DESCRIPTION OF THE FIGURES

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A is a graph that shows genes recurrently involved in gene fusionsin TCGA. Only genes involved in at least three gene fusions acrossdifferent samples are displayed.

FIGS. 1B-1, 1B-2, 1B-3 and 1B-4 show an FGFR3-TACC3 gene fusionidentified by whole transcriptome sequencing of GSCs. 76 split-reads(SEQ ID NOS: 2-77, respectively) are shown aligning on the breakpoint.The predicted reading frame at the breakpoint is shown at the top (FGFR3nucleotide sequence (left) and TACC3 nucleotide sequence (right); SEQ IDNO: 1) with FGFR3 sequences below the predicted reading frame (left) andTACC3 (right). The putative amino acid sequence (SEQ ID NO: 78)corresponding to SEQ ID NO: 1 is shown above the predicted readingframe.

FIG. 1C shows an FGFR3-TACC3 gene fusion identified by wholetranscriptome sequencing of GSCs. On the left, FGFR3-TACC3-specific PCRfrom cDNA derived from GSCs and GBM is shown. On the right, Sangersequencing chromatogram shows the reading frame at the breakpoint (SEQID NO: 80) and putative translation of the fusion protein (SEQ ID NO:85) in the positive samples.

FIG. 1D shows an FGFR3-TACC3 gene fusion identified by wholetranscriptome sequencing of GSCs. Amino acid sequence of the FGFR3-TACC3protein is shown (SEQ ID NO: 79). Residues corresponding to FGFR3 or toTACC3 (underlined) are shown. The fusion protein joins the tyrosinekinase domain of FGFR3 to the TACC domain of TACC3.

FIG. 1E shows an FGFR3-TACC3 gene fusion identified by wholetranscriptome sequencing of GSCs. Genomic fusion of FGFR3 exon 17 withintron 7 of TACC3 is shown. In the fused mRNA, exon 16 of FGFR3 isspliced 5′ to exon 8 of TACC3. Filled arrows indicate the position ofthe fusion-genome primers, which generate fusion-specific PCR productsin GSC-1123 and GBM-1123.

FIG. 2A shows recurrent gene fusions between FGFR and TACC genes in GBM.Specifically, FGFR3-TACC3 gene fusions are shown that were identified byexome sequencing analysis. Split-reads are shown aligning the genomicbreakpoints of FGFR3 and TACC3 genes in the four TCGA GBM samples. ForTCGA-27-1835, SEQ ID NO: 95 shows the reading frame at the breakpoint(bold), while SEQ ID NOS: 96-107, respectively, show alignments of thegenomic breakpoints. For TCGA-19-5958, SEQ ID NO: 108 shows the readingframe at the breakpoint (bold), while SEQ ID NOS: 109-111, respectively,show alignments of the genomic breakpoints. For TCGA-06-6390, SEQ ID NO:112 shows the reading frame at the breakpoint (bold), while SEQ ID NOS:113-131, respectively, show alignments of the genomic breakpoints. ForTCGA-12-0826, SEQ ID NO: 132 shows the reading frame at the breakpoint(bold), while SEQ ID NOS: 133-145, respectively, show alignments of thegenomic breakpoints.

FIG. 2B shows recurrent gene fusions between FGFR and TACC genes in GBM.On the left, a gel of FGFR-TACC-specific PCR is shown for FGFR3-TACC3from a GBM cDNA sample. On the right, Sanger sequencing chromatogramsshow the reading frame at the breakpoint (SEQ ID NO: 81) and putativetranslation of the fusion protein (SEQ ID NO: 86) in the positivesamples.

FIG. 2C shows recurrent gene fusions between FGFR and TACC genes in GBM.On the left, a gel of FGFR-TACC-specific PCR is shown for FGFR3-TACC3from a GBM cDNA sample. On the right, Sanger sequencing chromatogramsshow the reading frame at the breakpoint (SEQ ID NO: 82) and putativetranslation of the fusion protein (SEQ ID NO: 87) in the positivesamples.

FIG. 2D shows recurrent gene fusions between FGFR and TACC genes in GBM.Co-outlier expression of FGFR3 and TACC3 in four GBM tumors fromAtlas-TCGA is shown in the plot.

FIG. 2E shows recurrent gene fusions between FGFR and TACC genes in GBM.CNV analysis shows micro-amplifications of the rearranged portions ofthe FGFR3 and TACC3 genes in the same four Atlas-TCGA GBM samples.

FIG. 2F shows recurrent gene fusions between FGFR and TACC genes in GBM.On the left, a gel of FGFR-TACC-specific PCR is shown for FGFR1-TACC1from a GBM cDNA sample. On the right, Sanger sequencing chromatogramsshow the reading frame at the breakpoint (SEQ ID NO: 83) and putativetranslation of the fusion protein (SEQ ID NO: 88) in the positivesamples.

FIG. 2G shows recurrent gene fusions between FGFR and TACC genes in GBM.On the left, a gel of FGFR-TACC-specific PCR is shown for FGFR3-TACC3from a GBM cDNA sample. On the right, Sanger sequencing chromatogramsshow the reading frame at the breakpoint (SEQ ID NO: 84) and putativetranslation of the fusion protein (SEQ ID NO: 89) in the positivesamples.

FIG. 3A shows transforming activity of FGFR-TACC fusion proteins.FGFR1-TACC1 and FGFR3-TACC3 induce anchorage-independent growth in Rat1Afibroblasts. The number of soft agar colonies was scored from triplicatesamples 14 days after plating. Representative microphotographs areshown.

FIG. 3B are photomicrographs showing of immunofluorescence staining oftumors from mice injected with Ink4A;Arf−/− astrocytes expressingFGFR3-TACC3 showing positivity for glioma-specific (Nestin, Oig2 andGFAP) and proliferation markers (Ki67 and pHH3). Sub-cutaneous tumorswere generated by Ink4A;Arf−/− astrocytes expressing FGFR-TACC fusions.

FIG. 3C shows Kaplan-Meier survival curves of mice injectedintracranially with pTomo-shp53 (n=8) or pTomo-EGFRvIII-shp53 (n=7)(green line; “light grey” in black and white image) andpTomo-FGFR3-TACC3-shp53 (n=8, red line; “dark grey” in black and whiteimage). Points on the curves indicate deaths (log-rank test, p=0.00001,pTomo-shp53 vs. pTomo-FGFR3-TACC3-shp53).

FIG. 3D shows representative photomicrographs of Hematoxylin and Eosinstaining of advanced FGFR3-TACC3-shp53 generated tumors showinghistological features of high-grade glioma. Of note is the high degreeof infiltration of the normal brain by the tumor cellsImmunofluorescence staining shows that glioma and stem cell markers(Nestin, Olig2 and GFAP), the proliferation markers (Ki67 and pHH3) andthe FGFR3-TACC3 protein are widely expressed in the FGFR3-TACC3-shp53brain tumors. F1-T1: FGFR1-TACC1; F3-T3: FGFR3-TACC3; F3-T3-K508M:FGFR3-TACC3-K508M.

FIG. 4A shows that FGFR3-TACC3 localizes to spindle poles, delaysmitotic progression and induces chromosome segregation defects andaneuploidy Constitutive auto-phosphorylation of FGFR3-TACC3 fusion.Ink4A;Arf−/− astrocytes transduced with empty lentivirus or a lentivirusexpressing FGFR3-TACC3 or FGFR3-TACC3-K508M were left untreated (0) ortreated with 100 nM of the FGFR inhibitor PD173074 for the indicatedtimes. Phospho-proteins and total proteins were analyzed by Western blotusing the indicated antibodies.

FIG. 4B shows that FGFR3-TACC3 localizes to spindle poles, delaysmitotic progression and induces chromosome segregation defects.Photomicrographs are shown of confocal microscopy analysis ofFGFR3-TACC3 in Ink4A;Arf−/− astrocytes. Maximum intensity projection ofz-stacked images shows FGFR3-TACC3 (red; “dark grey” in black and whiteimage) coating the spindle poles of a representative mitotic cell (upperpanels). In telophase (lower panels) FGFR3-TACC3 localizes to themid-body. α-tubulin (green; “grey” in black and white image), DNA (DAPI,blue; “light grey” in black and white image).

FIG. 4C shows representative fluorescence video-microscopy for cellstransduced with vector or FGFR3-TACC3.

FIG. 4D shows a Box and Whisker plot representing the analysis of thetime from nuclear envelope breakdown (NEB) to anaphase onset and fromNEB to nuclear envelope reconstitution (NER). The duration of mitosiswas measured by following 50 mitoses for each condition by time-lapsemicroscopy.

FIG. 4E shows that FGFR3-TACC3 localizes to spindle poles, delaysmitotic progression and induces chromosome segregation defects.Representative images are shown of cells with chromosome missegregation.Arrows point to chromosome misalignments, lagging chromosomes, andchromosome bridges.

FIG. 4F shows quantitative analysis of segregation defects in Rat1Aexpressing FGFR1-TACC1 and FGFR3-TACC3. F3-T3: FGFR3-TACC3; F3-T3-K508M:FGFR3-TACC3-K508M.

FIG. 5A shows karyotype analysis of Rat1A cells transduced with control,FGFR3, TACC3 or FGFR3-TACC3 expressing lentivirus. Distribution ofchromosome counts of cells arrested in mitosis and analyzed forkaryotypes using DAPI. Chromosomes were counted in 100 metaphase cellsfor each condition to determine the ploidy and the diversity ofchromosome counts within the cell population. FGFR3-TACC3 fusion inducesaneuploidy.

FIG. 5B shows representative karyotypes and FIG. 5C shows distributionof chromosome counts of human astrocytes transduced with control orFGFR3-TACC3 expressing lentivirus. Chromosomes were counted in 100metaphase cells for each condition to determine the ploidy and thediversity of chromosome counts within the cell population.

FIG. 5D shows quantitative analysis of chromosome number in 100metaphase cells for each condition to determine the ploidy and thediversity of chromosome counts within the cell population. (n=3independent experiments).

FIG. 6A shows inhibition of FGFR-TK activity corrects the aneuploidyinitiated by FGFR3-TACC3. The upper panel is a karyotype analysis ofRat1A cells transduced with control or FGFR3-TACC3 lentivirus andtreated with vehicle (DMSO) or PD173470 (100 nM) for five days. Thelower panel shows the ploidy and the diversity of chromosome countswithin the cell population were determined by quantitative analysis ofchromosome number in 100 metaphase cells for each condition.

FIG. 6B shows inhibition of FGFR-TK activity corrects the aneuploidyinitiated by FGFR3-TACC3. Correction of premature sister chromatidseparation (PMSCS) by PD173470 in cells expressing FGFR3-TACC3. Panelsshow representative metaphase spreads. DNA was stained by DAPI. FIG. 6Cshows quantitative analysis of metaphases with loss of sister chromatidcohesion (n=3; p=0.001, FGFR3-TACC3 treated with DMSO vs. FGFR3-TACC3treated with PD173470).

FIG. 7A shows inhibition of FGFR-TK activity suppresses tumor growthinitiated by FGFR3-TACC3. Growth rate of Rat1A transduced with theindicated lentiviruses and treated for three days with increasingconcentrations of PD173074. Cell growth was determined by the MTT assay.Data are presented as the means±standard error (n=4).

FIG. 7B shows the growth rate of GSC-1123 treated with PD173470 at theindicated concentrations for the indicated times. Cell growth wasdetermined by the MTT assay. Data are presented as the means±standarderror (n=4).

FIG. 7C shows the growth inhibitory effect of silencing FGFR3-TACC3fusion. At the left, parallel cultures of GSC-1123 cells were transducedin triplicate. Rat1A cells expressing FGFR3-TACC3 fusion were transducedwith lentivirus expressing a non-targeting shRNA (Ctr) or shRNAsequences targeting FGFR3 (sh2, sh3, sh4). Five days after infectioncells were plated at density of 2×10⁴ cells/well in triplicate and thenumber of trypan blue excluding cells was scored at the indicated times.Infection with lentivirus expressing sh-3 and sh-4, the most efficientFGFR3 silencing sequences reverted the growth rate of FGFR3-TACC3expressing cultures to levels comparable to those of Rat1A transducedwith empty vector. Values are the means±standard deviation (n=3). At theright sided figure, GSC-1123 cells were transduced with lentivirusexpressing a non-targeting shRNA (sh-Ctr) or lentivirus expressing sh-3and sh-4 sequences targeting FGFR3. Western Blot analysis was performedon parallel cultures using the FGFR3 antibody to the detect FGFT3-TACC3fusion protein. β-actin is shown as a control for loading.

FIG. 7D shows that the FGFR inhibitor PD173074 suppresses tumor growthof glioma sub-cutaneous xenografts generated by Ink4A;Arf−/− astrocytesexpressing FGFR3-TACC3. After tumor establishment (200-300 mm³, arrow)mice were treated with vehicle or PD173074 (50 mg/kg) for 14 days.Values are mean tumor volumes±standard error (n=7 mice per group).

FIG. 7E is a Kaplan-Meier analysis of glioma-bearing mice followingorthotopic implantation of Ink4A;Arf−/− astrocytes transduced withFGFR3-TACC3. After tumor engraftment mice were treated with vehicle(n=9) or AZD4547 (50 mg/kg) (n=7) for 20 days (p=0.001).

FIG. 8 shows a schematic of the TX-Fuse pipeline for the identificationof fusion transcripts from RNA-Seq data generated from nine GSCcultures. The continued figure shows a schematic of the Exome-Fusepipeline for the identification of gene fusion rearrangements from DNAexome sequences of 84 GBM TCGA tumor samples.

FIGS. 9A-D shows the validation of fusion transcripts identified byRNA-seq of nine GSCs. Sanger sequencing chromatograms show the readingframes at the breakpoint and putative translation of the fusion proteinsin the positive samples (right side). The left side shows gels of RT-PCRconducted. (A) POLR2A-WRAP53. DNA sequence disclosed as SEQ ID NO: 319and protein sequence disclosed as SEQ ID NO: 320. (B) CAPZB-UBR4. DNAsequence disclosed as SEQ ID NO: 321 and protein sequence disclosed asSEQ ID NO: 322. (C) ST8SIA4-PAM. DNA sequence disclosed as SEQ ID NO:323 and protein sequence disclosed as SEQ ID NO: 324. (D) PIGU-NCOA6.DNA sequence disclosed as SEQ ID NO: 325 and protein sequence disclosedas SEQ ID NO: 326.

FIGS. 9E-1, 9E-2, 9E-3, 9E-4, 9E-5, 9E-6, 9E-7, and 9E-8 show the fusiontranscripts identified by whole transcriptome sequencing of nine GSCs.54 split-reads (SEQ ID NOS 329-382, respectively, in order ofappearance) are shown aligning on the breakpoint of the POLR2A-WRAP53fusion (SEQ ID NO: 327). The predicted reading frame at the breakpointis shown at the top with POLR2A sequences in red (left) and WRAP53 inblue (right). Protein sequence disclosed as SEQ ID NO: 328. On thecontinued page, 48 split-reads (SEQ ID NOS 385-432, respectively, inorder of appearance) are shown aligning on the breakpoint of theCAPZB-UBR4 fusion (SEQ ID NO: 383). The predicted reading frame at thebreakpoint is shown at the top with CAPZB sequences in red (left) andUBR4 in blue (right). Protein sequence disclosed as SEQ ID NO: 384. Onthe continued page after, 29 split-reads (SEQ ID NOS 435-463,respectively, in order of appearance) are shown aligning on thebreakpoint of the ST8SIA4-PAM fusion (SEQ ID NO: 433). The predictedreading frame at the breakpoint is shown at the top with ST8SIA4sequences in red (left) and PAM in blue (right). Protein sequencedisclosed as SEQ ID NO: 434. On the subsequent continued page, 17split-reads (SEQ ID NOS 466-482, respectively, in order of appearance)are shown (top) aligning on the breakpoint of the PIGU-NCOA6 fusion (SEQID NO: 464). The predicted reading frame at the breakpoint is shown atthe top with PIGU sequences in red (left) and NCOA6 in blue (right).Protein sequence disclosed as SEQ ID NO: 465. Also (below), 6split-reads (SEQ ID NOS 485-490, respectively, in order of appearance)are shown aligning on the breakpoint of the IFNAR2-IL10RB fusion (SEQ IDNO: 483). The predicted reading frame at the breakpoint is shown at thetop with IFNAR2 sequences in red (left) and IL10RB in blue (right).Protein sequence disclosed as SEQ ID NO: 484.

FIG. 10A shows the analysis and validation of the expression of fusedtranscripts in GSCs and GBM sample. Expression measured by read depthfrom RNA-seq data. Light grey arcs indicate predicted components oftranscripts fused together. Overall read depth (blue; “grey” in blackand white image) and split insert depth (red; “dark grey” in black andwhite image) are depicted in the graph, with a 50-read increment and amaximum range of 1800 reads. Note the very high level of expression inthe regions of the genes implicated in the fusion events, particularlyfor FGFR3-TACC3.

FIG. 10B shows the analysis and validation of the expression of fusedtranscripts in GSCs and GBM sample. Top panel, qRT-PCR showing the veryhigh expression of FGFR3 and TACC3 mRNA sequences included in theFGFR3-TACC3 fusion transcript in GSC-1123. Bottom panel, for comparisonthe expression of sequences of WRAP53 mRNA included in the POLR2A-WRAP53fusion in GSC-0114 is also shown.

FIG. 10C shows the expression of the FGFR3-TACC3 protein in GSC-1123 andGBM-1123. Western blot analysis with a monoclonal antibody, whichrecognizes the N-terminal region of human FGFR3 shows expression of a˜150 kD protein in GSC-1123 but not in the GSC cultures GSC-0331 andGSC-0114, which lack the FGFR3-TACC3 rearrangement.

FIG. 10D shows the analysis and validation of the expression of fusedtranscripts in GSCs and GBM sample Immunostaining analysis with theFGFR3 antibody of the tumor GBM-1123 (top panel) and a GBM tumor lackingthe FGFR3-TACC3 rearrangement. FGFR3 (red; “light grey” in black andwhite image), DNA (DAPI, blue; “grey” in black and white image). Thepictures were taken at low (left) and high (right) magnification.

FIGS. 10E-1, 10E-2, 10E-3, 10E-4, 10E-5, and 10E-6 shows MS/MS analysisof the ˜150 kD fusion protein immunoprecipitated by the monoclonalanti-FGFR3 antibody from GSC-1123, identifying three unique peptidesmapping to the FGFR3 (FGFR3 Peptide 1 (SEQ ID NO: 492), 2 (SEQ ID NO:493), and 3 (SEQ ID NO: 494)) and three peptides mapping to theC-terminal region of TACC3 (TACC Peptide 1 (SEQ ID NO: 156), 2 (SEQ IDNO: 157), and 3 (SEQ ID NO: 491)).

FIGS. 11A-C shows Rat1A cells transduced with control lentivirus orlentivirus expressing FGFR3, TACC3, FGFR3-TACC3 (FIG. 11A) that wereanalyzed by Western blot with an antibody recognizing the N-terminus ofFGFR3 (included in the FGFR3-TACC3 fusion protein) or the N-terminus ofTACC3 (not included in the FGFR3-TACC3 fusion protein). FIG. 11B showsquantitative Western blot analysis of endogenous FGFR3-TACC3 in GSC-1123compared with lentivirally expressed FGFR3-TACC3 in Rat1A. FIG. 11Cshows Western blot analysis of FGFR3-TACC3 and FGFR3-TACC3-K508M inRat1A. α-tubulin is shown as a control for loading.

FIGS. 11D-F shows expression analyses of FGFR3-TACC3 fusion construct(FIG. 11D) FGFR3 immunostaining of GBM-1123 (left, upper panel),BTSC1123 (right, upper panel), mouse GBM induced by FGFR3-TACC3expressing lentivirus (left, lower panel), and sub-cutaneous xenograftof mouse astrocytes transformed by FGFR3-TACC3 fusion (right, lowerpanel); FGFR3-TACC3, red (“light grey” in black and white image); DNA(DAPI), blue (“grey” in black and white image). FIG. 11E showsquantification of FGFR3-TACC3 positive cells in the tumors and culturesof cells shown in FIG. 11D. FIG. 11F shows a quantitative Western blotanalysis of ectopic FGFR3-TACC3 fusion protein in mouse astrocytes andFGFR3-TACC3 induced mouse GBM (mGBM-15 and mGBM-17) compared with theendogenous expression in GBM1123. β-actin is shown as a control forloading. F3-T3: FGFR3-TACC3. α-tubulin or β-actin is shown as a controlfor loading.

FIG. 12A shows a western blot. Ink4A;Arf−/− astrocytes transduced withempty lentivirus or a lentivirus expressing FGFR3-TACC3 were starved ofmitogens and left untreated (time 0) or treated with FGF-2 atconcentration of 50 ng/ml for the indicated times. Phospho-proteins andtotal proteins were analyzed by Western blot using the indicatedantibodies. α-tubulin is shown as a control for loading.

FIG. 12B show western blots. Ink4A;Arf−/− astrocytes transduced withempty lentivirus or a lentivirus expressing FGFR3-TACC3 orFGFR3-TACC3-K508M were starved of mitogens and left untreated (time 0)or treated for 10 min with FGF-1 at the indicated concentrations.Phospho-proteins and total proteins were analyzed by Western blot usingthe indicated antibodies. β-actin is shown as a control for loading.

FIG. 12C show western blots. Ink4A;Arf−/− astrocytes transduced withempty lentivirus or a lentivirus expressing FGFR3-TACC3 orFGFR3-TACC3-K508M were starved of mitogens and left untreated (time 0)or treated for 10 min with FGF-8 at the indicated concentrations.Phospho-proteins and total proteins were analyzed by Western blot usingthe indicated antibodies. β-actin is shown as a control for loading.

FIGS. 12D-F shows mitotic localization of FGFR3-TACC3 fusion protein.FIG. 12D shows maximum intensity projection confocal image of arepresentative FGFR3-TACC3 expressing Ink4A;Arf−/− mouse astrocyte atmetaphase immunostained using the FGFR3 antibody (red; “dark grey” inblack and white image). FGFR3-TACC3 displays asymmetric localization ontop of one spindle pole. FIG. 12E shows maximum intensity projectionconfocal image of a representative TACC3 expressing Ink4A;Arf−/− mouseastrocyte at metaphase immunostained with the TACC3 antibody (red;(“dark grey” in black and white image). TACC3 staining coincides withthe spindle microtubules. FIG. 12F shows maximum intensity projectionconfocal image of a representative FGFR3 expressing Ink4A;Arf−/− mouseastrocyte at metaphase immunostained with the FGFR3 antibody (red;(“dark grey” in black and white image). FGFR3 does not show a specificstaining pattern in mitosis. Cells were co-immunostained using α-tubulin(green; (“light grey” in black and white image) to visualize the mitoticspindle. DNA was counterstained with DAPI (blue; (“grey” in black andwhite image). Images were acquired at 0.250 μm intervals. Endogenouslevels of FGFR3 or TACC3 were undetectable under the appliedexperimental conditions. F3-T3: FGFR3-TACC3.

FIG. 13A shows that the FGFR3-TACC3 protein induces chromosomalmissegregation, chromatid cohesion defects and defective spindlecheckpoint. Quantitative analysis of metaphase spreads for chromosomesegregation defects in Ink4A;ARF−/− astrocytes expressing vector controlor FGFR3-TACC3 (upper panel). Microscope imaging analysis of chromosomesegregation defects in Ink4A;Arf−/− astrocytes expressing FGFR3-TACC3 orvector control. Representative images of cells with chromosomemissegregation. Arrows point to chromosome misalignments, laggingchromosomes and chromosome bridges.

FIGS. 13B-D shows representative images of premature sister chromatidseparation (PMSCS) in Ink4A;Arf−/− astrocytes (FIG. 13B) and Rat1A cells(FIG. 13C) expressing FGFR3-TACC3. Left, panels show representativemetaphase spreads. Right, quantitative analysis of metaphases with lossof sister chromatid cohesion. The number of mitosis with PMSCS inInk4A;Arf−/− astrocytes was scored in at least 100 metaphases for eachcondition in three independent experiments. The number of mitosis withPMSCS was scored in triplicate samples of Rat1A cells. FIG. 13D is agraph showing nocodazole was added for the indicated durations toRat1A-H2B-GFP cells transduced with the specified lentiviruses. Themitotic index at each time point was determined by quantitating theH2B-GFP-positive cells in mitosis at each time point. Data are presentedas average and standard deviation (n=3). F3-T3: FGFR3-TACC3.

FIGS. 14A-B shows growth curves of human primary astrocytes transducedwith lentivirus expressing FGFR3-TACC3 fusion or the empty vector. Ananalysis was conducted of FGFR3-TACC3 fusion mediated growth alterationand specific effect of RTK inhibitors on cells carrying FGFR-TACCfusions. FIG. 14A is a graph that shows cell proliferation of humanprimary astrocytes transduced with lentivirus expressing FGFR3-TACC3fusion or the empty vector was determined by the MTT assay 7 days afterinfection (passage 1). Values are the means±standard deviation (n=4).p-value: 0.0033. FIG. 14B is a graph that shows cell proliferation ofhuman primary astrocytes transduced with lentivirus expressingFGFR3-TACC3 fusion or the empty vector was determined by the MTT assaysix weeks after the infection (passage 10). Values are themeans±standard deviation (n=4). p-value: 0.0018.

FIGS. 14C-D shows specific growth inhibitory effect by FGFR inhibitorson FGFR-TACC fusion expressing cells. Cell growth was determined by MTTassay. Rat1A cells transduced with the indicated lentivirus were treatedfor three days with BGJ398 (FIG. 14C) or AZD4547 (FIG. 14D) at theindicated concentration. Values are the means±standard error (n=4).

FIG. 14E shows the growth inhibitory effect of silencing FGFR3-TACC3fusion. (left) GSC-1123 cells were transduced in triplicate withlentivirus expressing a non-targeting shRNA (Ctr) or lentivirusexpressing sh-3 and sh-4 sequences targeting FGFR3. Five days afterinfection cells were plated at density of 2×10⁴ cells/well in triplicateand the number of trypan blue excluding cells was scored at theindicated times. Values are the means±standard deviation (n=3). (right)Western Blot analysis was performed on parallel cultures collected fivedays after infection using the FGFR3 antibody to the detect FGFT3-TACC3fusion protein. β-actin is shown as a control for loading. (**:p-value=<0.005; ***: p-value=<0.0001).

FIG. 15 shows a survival plot of cells treated with PD173074,NVP-BGJ398, or AZD4547.

FIG. 16 shows an FGFR3-TACC3 gene fusion identified by wholetranscriptome sequencing of GSCs. The histogram describes the absolutefrequency of each forward and reverse sequence read spanning thebreakpoint.

FIG. 17 shows transforming activity of FGFR3-TACC3. FGFR3-TACC3 inducesanchorage-independent growth in Rat1A fibroblasts (top panels) and atransformed phenotype in Ink4A;Arf−/− primary astrocytes (bottompanels).

FIG. 18 shows transforming activity of FGFR3-TACC3. Kaplan-Meiersurvival curves of mice injected intracranially with pTomo-shp53 (n=8),pTomo-FGFR3-TACC3-shp53 (n=8) and pTomo-EGFRvIII-shp53 (n=7) are shown.Points on the curves indicate deaths (log-rank test, p=0.025,pTomo-shp53 vs. pTomo-FGFR3-TACC3-shp53).

FIG. 19 shows that inhibition of FGFR-TK activity corrects theaneuploidy and suppresses tumor growth initiated by FGFR3-TACC3.Short-term growth inhibition assays are shown of Rat1A transduced withthe indicated lentivirus and treated with PD173470 at the indicatedconcentrations. Cells were treated for three days. Cell viability wasdetermined by the MTT assay. Error bars show means±standard error (n=4).

FIG. 20 is a growth inhibition assay of human astrocytes transduced withthe indicated lentivirus and treated for four days with PD173470 at theindicated concentration. Cell viability was determined by the MTT assay.Error bars show means±standard error (n=4).

FIG. 21 is a graph showing a growth inhibition assay of human astrocytestransduced with the indicated lentivirus and treated for four days withPD173470 at the indicated concentration. Cell viability was determinedby the MTT assay. Error bars show means±standard error (n=4).

FIG. 22 shows graphs of the survival of Rat1A cells in short-term growthinhibition assays. (Top graph) Rat1A cells were transduced with theindicated ptomo constructs and treated with PD173074 at the indicatedconcentrations. Cells were treated for three days. Cell viability wasdetermined by the MTT assay. Error bars show means±standard error (n=4).In the bottom panel, a western blot photograph is shown.

FIG. 23 shows that inhibition of FGFR-TK activity corrects theaneuploidy and suppresses tumor growth initiated by FGFR3-TACC3. A plotis shown of karyotype analysis of Rat1A cells transduced with control orFGFR3-TACC3 lentivirus and treated with vehicle (DMSO) or PD173470 (100nM) for five days.

FIG. 24 shows Survival of glioma-bearing mice was tracked followingintracranial implantation of Ink4A;Arf−/− astrocytes transduced withFGFR3-TACC3. After tumor engraftment mice were treated with vehicle orAZD4547 (50 mg/kg) for 20 days (vehicle, n=7; AZD4547, n=6; p=0.001).

FIG. 25 shows the position of the peptides from FIGS. 10E1-10E6 in theamino acid sequence of the FGFR3-TACC3 fusion protein (SEQ ID NO: 79),which are highlighted in pink (FGFR3; underlined) and blue (TACC3;dotted lines).

FIG. 26 shows Kaplan-Meier analysis of IDH mutant and FGFR3-TACC3positive human GBM. Log rank test p-value: 0.0169.

FIGS. 27A-B are pictures that shows tumor xenografts that were inducedfollowing sub-cutaneous injection of Ink4A;Arf−/− mouse astrocytestransduced with lentivirus expressing FGFR3-TACC3 (upper panel A, rightflank) or FGFR1-TACC1 (lower panel B, right flank) fusion, but not withthe empty vector (upper panel, left flank) or FGFR3-TACC3 carrying aK508M mutation in the kinase domain (FGFR3-TACC3-K508M; lower panel,left flank).

FIG. 28 shows constitutive auto-phosphorylation of FGFR3-TACC3 fusion.BTSC derived from FGFR3-TACC3 or RasV12 induced mouse GBM were leftuntreated or treated with 500 nM PD173074 for the indicated times.Phospho-proteins and total proteins were analyzed by Western blot usingthe indicated antibodies. β-actin is shown as a control for loading.

FIG. 29 shows Z-stacked confocal images of the representativeFGFR3-TACC3 expressing Ink4A;Arf−/− mouse astrocyte shown as a maximumintensity projection. Cells were immunostained using FGFR3 (red; “darkgrey” in black and white image) and α-tubulin (green; (“light grey” inblack and white image). DNA was counterstained with DAPI (blue; (“grey”in black and white image). Images were acquired at 0.250 μm intervals.Coordinates of the image series are indicated. F3-T3: FGFR3-TACC3.

FIG. 30 shows examples of SKY karyotype analysis painting two differentcells from the same culture of GSC-1123, illustrating the ongoing CINand aneuploidy. Details of the karyotype analysis of 20 cells arereported in Table 6.

FIGS. 31-1, 31-2, and 31-3 are a graphical representation of segmentedCNVs data visualized using the Integrated Genomic Viewers software.Three bladder Urothelial Carcinoma harbor FGFR3-TACC3 gene fusions(black box). Red indicates amplification (A), blue indicates deletion(D).

FIGS. 32-1, 32-2, and 32-3 are a graphical representation of segmentedCNVs data visualized using the Integrated Genomic Viewers software. OneBreast Carcinoma harbors FGFR3-TACC3 gene fusions (black box). Redindicates amplification (A), blue indicates deletion (D).

FIGS. 33-1, 33-2, and 33-3 are a graphical representation of segmentedCNVs data visualized using the Integrated Genomic Viewers software. OneColorectal Carcinoma harbors FGFR3-TACC3 gene fusions (black box). Redindicates amplification (A), blue indicates deletion (D).

FIGS. 34-1, 34-2, and 34-3 are a graphical representation of segmentedCNVs data visualized using the Integrated Genomic Viewers software. OneLung Squamous Cell Carcinoma harbors FGFR3-TACC3 gene fusions (blackbox). Red indicates amplification (A), blue indicates deletion (D).

FIGS. 35-1, 35-2, and 35-3 are a graphical representation of segmentedCNVs data visualized using the Integrated Genomic Viewers software. OneHead and Neck Squamous Cell Carcinoma harbors FGFR3-TACC3 gene fusions(black box). Red indicates amplification (A), blue indicates deletion(D).

FIG. 36 shows the structure of FGFR-TACC gene fusions identified byRT-PCR-Sanger sequencing (see also SEQ ID NOs: 530-547). PredictedFGFR-TACC fusion proteins encoded by the transcripts identified byRT-PCR. Regions corresponding to FGFR3 or TACC3 are shown in red orblue, respectively. FGFR1 and TACC1 corresponding regions are shown inyellow and green. On the left are indicated the FGFR and TACC exonsjoined in the fused mRNA; the presence of TACC3 introns is also reportedwhen they are spliced in the fusion cDNA. On the right, the number ofpatients harboring the corresponding fusion variant is indicated. Thenovel transcripts discovered in this study are highlighted in red. Blackarrows indicate the position of the primers used for the FGFR-TACCfusions screening.

FIGS. 37A-H show the identification and immunostaining ofFGFR3-TACC3-positive tumors. Results from RT-PCR screening inrepresentative samples from the Pitié-Salpêtrière Hospital (A, C) andthe Besta (B, D) datasets. M, DNA ladder. Schematic representation ofthe FGFR3-TACC3 fusion transcripts identified in samples GBM-4620 (C)and GBM-021 (D). The junction sequences on the mRNA (GBM-4620 (C) SEQ IDNO: 515; GBM-021 (D) SEQ ID NO: 517) and the reading frame andtranslation (GBM-4620 (C) SEQ ID NO: 516; GBM-021 (D) SEQ ID NO: 518) atthe breakpoint are reported. Representative microphotographs of H&E andFGFR3 immunostaining in the FGFR3-TACC3 positive samples GBM-4620 (E)and GBM-021 (F) and two FGFR3-TACC3 negative samples (panels G and H);a, H&E, 10× magnification; b, H&E, 40× magnification; c, FGFR3, 10×magnification; d, FGFR3, 40× magnification.

FIGS. 38A-D show pre-clinical evaluation of FGFR3-TACC3 inhibition byJNJ-42756493. (A) Mouse astrocytes expressing FGFR3-TACC3 (F3T3),FGFR3-TACC3-KD (F3T3-KD) or the empty vector (Vector) were treated withthe indicated concentration of JNJ-42756493. Cell viability wasdetermined by the MTT assay. Error bars show mean±SEM (n=6). (B)Survival analysis of GIC28 1123 treated with JNJ-42756493. (C) TheFGFR-TK inhibitor JNJ-42756493 suppresses tumor growth of subcutaneoustumors generated by GIC-1123. After tumor establishment (arrow) micewere treated with vehicle or JNJ-42756493 (12 mg/kg) for 14 days. Valuesare mean tumor volumes±SD, (n=9 mice per group). P-value of the slopecalculated from the treatment starting point (arrow) is 0.04. (D)Photograph showing the tumors dissected from vehicle or JNJ-42756493treated mice after two weeks of treatment.

FIGS. 39A-G show baseline and post-treatment Magnetic Resonance Imaging(MRI) of patients treated with JNJ-42756493. Patient 1 (Panels A-D). (A)Post-gadolinium T1 weighted images show the target lesion on the rightparietal lobe. The interval (days) from the beginning of follow-up isindicated above each MRI. (B) Analysis of sum of product diameters (SPD)before and during the anti-FGFR treatment (RANO criteria). (C) Analysisof tumor volume (cm3) before and during the anti-FGFR treatment. Duringanti-FGFR treatment a stabilization of the tumor was observed accordingto RANO criteria and volumetry. (D) Perfusion images at baseline andafter 20 days of anti-FGFR treatment. rCBV (relative cerebral bloodvolume). Post-gadolinium T1 weighted images with color overlay of rCBVare shown. Patient 2 (Panels E-G). (E) Two different MRI slice levels ofsuperior and middle part of the lesion are presented. (F) Analysis ofsum of product diameters (SPD) before and during the anti-FGFRtreatment. During the anti-FGFR treatment a reduction of 22% of tumorsize was observed. (G) Volumetric evaluation showed a 28% tumorreduction. Vertical red arrow indicates the start of anti-FGFR treatment(baseline).

FIG. 40 shows the genomic PCR images and Sanger sequences of FGFR3-TACC3genomic breakpoints. Fusion specific PCR products and Sanger sequencingchromatograms showing the FGFR3-TACC3 genomic breakpoints (Sample #4451SEQ ID NO:519; Sample #OPK-14 SEQ ID NO: 520; Sample #MB-22 SEQ ID NO:521; Sample #3048 SEQ ID NO: 522; Sample #4373 SEQ ID NO: 523; Sample#4867 SEQ ID NO: 524; Sample #3808 SEQ ID NO: 525; Sample #27-1835 SEQID NO: 526; Sample #06-6390 SEQ ID NO: 527). The genomic sequencescorresponding to FGFR3 and TACC3 are indicated in red or blue,respectively. M, DNA ladder; C−, Negative Control.

FIG. 41 shows schematics of FGFR3-TACC3 genomic breakpoints. Schematicrepresentation of the genomic fusions between FGFR3 and TACC3 comparedto the corresponding mRNA. In red and blue are reported the regionsbelonging to FGFR3 and TACC3, respectively. The genomic breakpointcoordinates, according to the genome build GRCh37/hg19, are indicatedabove each fusion gene.

FIGS. 42A-B show evaluation of the expression of FGFR3-TACC3 fusionelements. (A) Microphotographs of immunofluorescence staining of arepresentative GBM harboring FGFR3-TACC3 fusion using antibodies thatrecognize the N- and C-termini of FGFR3 (FGFR3-N, FGFR3-C) and TACC3(TACC3-N, TACC3-C), red. Nuclei are counterstained with DAPI, blue. (B)Quantitative RT-PCR of four representative GBM carrying FGFR3-TACC3fusion and three negative controls using primer pairs that amplify FGFR3and TACC3 regions included in or excluded from the fusion transcripts,as indicated in the diagram. OAW28: ovarian cystoadenocarcinoma cellline harboring wild type FGFR3 and TACC3 genes; GBM55 and GBM0822: GBMharboring wild type FGFR3 and TACC3 genes; GBM3808; GBM1133; GBM0826;GBM3048: GBM harboring FGFR3-TACC3 (F3-T3) fusion. Error bars are SD oftriplicate samples.

FIGS. 43A-C show the FGFR3-TACC3 fusion gene and protein are retained inrecurrent GBM. (A) FGFR3-TACC3 fusion specific RT-PCR product fromuntreated and recurrent GBM from patient #3124. (B) Sanger sequencingchromatogram showing the identical reading frame at the breakpoint (SEQID NO: 517) and the putative translation of the fusion protein (SEQ IDNO: 86) in the untreated and recurrent tumor from the same patient. Thefused exons at mRNA level are shown. Regions corresponding to FGFR3 andTACC3 are indicated in red and blue, respectively. T, threonine; S,serine; D, aspartic acid; V, valine; K, lysine; A, alanine. (C)Representative microphotographs of FGFR3 immunofluorescence (IF)staining in both untreated and recurrent GBM. Blue staining, DAPI; Redstaining, FGFR3. 10× Magnification.

FIGS. 44A-B show PFS and OS of FGFR3-TACC3-positive glioma patients. (A)Kaplan-Meier curves in IDH wild-type glioma patients don't showsignificant differences in Progression Free Survival (PFS) betweenFGFR3-TACC3 positive (N=12, median PFS=11.20 months) and FGFR3-TACC3negative (N=274, Median PFS=12.27 months) (P=0.85). (B) Kaplan-Meiercurves in IDH wild-type glioma patients don't show significantdifferences in Overall Survival (OS) between FGFR3-TACC3 positive (N=12,Median OS=32.80 months) and FGFR3-TACC3 negative (N=326, Median OS=18.60months) (P=0.6). In red FGFR3-TACC3 positive patients, in greenFGFR3-TACC3 negative patients. Open circles represent censored patients.

FIG. 45 shows analysis of SNP6.0 arrays of GBM harboring CNVs of FGFR3and TACC3 genomic loci. CNVs of the FGFR3/TACC3 genomic loci in “gainlabeled” (LRR>0.2) TCGA samples. The CNA magnitudes (expressed as log 2ratio) were classified using simple thresholds: deletion (x<−1), loss(−1<x≦−0.2), gain (0.2≦x<1) or amplification (x>1). Gains are ingradient of red, loss in gradient of blue. Samples with uniformgains/amplification of FGFR3 and TACC3 lack FGFR3-TACC3 fusions. Samplesharboring FGFR3-TACC3 fusions (F3-T3) show microamplifications involvingthe first FGFR3 exons, which are spliced in the fusion gene.

DETAILED DESCRIPTION OF THE INVENTION

Glioblastoma multiformes (GBMs) are the most common form of brain tumorsin adults accounting for 12-15% of intracranial tumors and 50-60% ofprimary brain tumors. GBM is among the most lethal forms of humancancer. The history of successful targeted therapy of cancer largelycoincides with the inactivation of recurrent and oncogenic gene fusionsin hematological malignancies and recently in some types of epithelialcancer. GBM is among the most lethal and incurable forms of humancancer. Targeted therapies against common genetic alterations in GBMhave not changed the dismal clinical outcome of the disease, most likelybecause they have systematically failed to eradicate the truly addictingoncoprotein activities of GBM. Recurrent chromosomal rearrangementsresulting in the creation of oncogenic gene fusions have not been foundin GBM.

GBM is among the most difficult forms of cancer to treat in humans (1).So far, the therapeutic approaches that have been tested againstpotentially important oncogenic targets in GBM have met limited success(2-4). Recurrent chromosomal translocations leading to production ofoncogenic fusion proteins are viewed as initiating and addicting eventsin the pathogenesis of human cancer, thus providing the most desirablemolecular targets for cancer therapy (5, 6). Recurrent and oncogenicgene fusions have not been found in GBM. Chromosomal rearrangements arehallmarks of hematological malignancies but recently they have also beenuncovered in subsets of solid tumors (breast, prostate, lung andcolorectal carcinoma) (7, 8). Important and successful targetedtherapeutic interventions for patients whose tumors carry theserearrangements have stemmed from the discovery of functional genefusions, especially when the translocations involve kinase-coding genes(BCR-ABL, EML4-ALK) (9, 10).

A hallmark of GBM is rampant chromosomal instability (CIN), which leadsto aneuploidy (11). CIN and aneuploidy are early events in thepathogenesis of cancer (12). It has been suggested that geneticalterations targeting mitotic fidelity might be responsible formissegregation of chromosomes during mitosis, resulting in aneuploidy(13, 14).

Fibroblast growth factor receptors (FGFR) are transmembrane receptorsthat bind to members of the fibroblast growth factor family of proteins.The structure of the FGFRs consist of an extracellular ligand bindingdomain comprised of three Ig-like domains, a single transmembrane helixdomain, and an intracellular domain with tyrosine kinase activity(Johnson, D. E., Williams, E. T. Structural and functional diversity inthe FGF receptor multigene family. (1993) Adv. Cancer Res, 60:1-41).

Transforming acidic coiled-coiled protein (TACC) stabilize microtubulesduring mitosis by recruiting minispindles (Msps)/XMAP215 proteins tocentrosomes. TACCs have been implicated in cancer.

From a medical perspective, the FGFR-TACC fusions provide the first“bona-fide” oncogenically addictive gene fusions in GBM whoseidentification has long been overdue in this disease.

Beside GBM, which features the highest grade of malignancy among glioma(grade IV), lower grade glioma which include grade II and grade III area heterogeneous group of tumors in which specific molecular features areassociated with divergent clinical outcome. The majority of grade II-IIIglioma (but only a small subgroup of GBM) harbor mutations in IDH genes(IDH1 or IDH2), which confer a more favorable clinical outcome.Conversely, the absence of IDH mutations is associated with the worstprognosis (5).

Described herein is the identification of FGFR-TACC gene fusions (mostlyFGFR3-TACC3, and rarely FGFR1-TACC1) as the first example of highlyoncogenic and recurrent gene fusions in GBM. The FGFR-TACC fusions thathave been identified so far include the Tyrosine Kinase (TK) domain ofFGFR and the coiled-coil domain of TACC proteins, both necessary for theoncogenic function of FGFR-TACC fusions. FGFR3-TACC3 fusions have beenidentified in pediatric and adult glioma, bladder carcinoma, squamouslung carcinoma and head and neck carcinoma, thus establishing FGFR-TACCfusions as one of the chromosomal translocation most frequently foundacross multiple types of human cancers (6-15).

Here a screening method for FGFR-TACC fusions is reported that includesa RT-PCR assay designed to identify the known and novel FGFR3-TACC3fusion transcripts, followed by confirmation of the inframe breakpointby Sanger sequencing. Using this assay, a dataset of 584 GBM and 211grade II and grade III gliomas has been analyzed. It was determined thatbrain tumors harboring FGFR-TACC fusions manifest strong and homogeneousintra-tumor expression of the FGFR3 and TACC3 component invariablyincluded in the fusion protein, when analyzed by immunostaining. Asignificant clinical benefit following treatment with a specificinhibitor of FGFR-TK is reported in two GBM patients who harboredFGFR3-TACC3 rearrangement.

DNA and Amino Acid Manipulation Methods and Purification Thereof

The practice of aspects of the present invention can employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, e.g.,Molecular Cloning A Laboratory Manual, 3^(rd) Ed., ed. by Sambrook(2001), Fritsch and Maniatis (Cold Spring Harbor Laboratory Press:1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985);Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S.Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J.Higgins eds. 1984); Transcription and Translation (B. D. Hames & S. J.Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells and Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the series,Methods In Enzymology (Academic Press, Inc., N.Y.), specifically,Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987,Cold Spring Harbor Laboratory); Immunochemical Methods In Cell AndMolecular Biology (Caner and Walker, eds., Academic Press, London,1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir andC. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Allpatents, patent applications and references cited herein areincorporated by reference in their entireties.

One skilled in the art can obtain a protein in several ways, whichinclude, but are not limited to, isolating the protein via biochemicalmeans or expressing a nucleotide sequence encoding the protein ofinterest by genetic engineering methods.

A protein is encoded by a nucleic acid (including, for example, genomicDNA, complementary DNA (cDNA), synthetic DNA, as well as any form ofcorresponding RNA). For example, it can be encoded by a recombinantnucleic acid of a gene. The proteins of the invention can be obtainedfrom various sources and can be produced according to various techniquesknown in the art. For example, a nucleic acid that encodes a protein canbe obtained by screening DNA libraries, or by amplification from anatural source. A protein can be a fragment or portion thereof. Thenucleic acids encoding a protein can be produced via recombinant DNAtechnology and such recombinant nucleic acids can be prepared byconventional techniques, including chemical synthesis, geneticengineering, enzymatic techniques, or a combination thereof. Forexample, a fusion protein of the invention comprises a tyrosine kinasedomain of an FGFR protein fused to a polypeptide that constitutivelyactivates the tyrosine kinase domain of the FGFR protein. For example, afusion protein of the invention comprises a transforming acidiccoiled-coil (TACC) domain fused to a polypeptide with a tyrosine kinasedomain, wherein the TACC domain constitutively activates the tyrosinekinase domain. An example of a FGFR1-TACC1 polypeptide has the aminoacid sequence shown in SEQ ID NO: 150. An example of a FGFR3-TACC3protein is the polypeptide encoded by the nucleic acid having thenucleotide sequence shown in SEQ ID NOs: 94, 530, 531, 532, 533, 534,535, 536, 537, or 538. Examples of a FGFR3-TACC3 polypeptide has theamino acid sequence shown in SEQ ID NO: 79, 158, 159, 160, 161, 539,540, 541, 542, 543, 544, 545, 546, or 547.

The Genbank ID for the FGFR3 gene is 2261. Three isoforms are listed forFGFGR3, e.g., having Genebank Accession Nos. NP_(—)000133 (correspondingnucleotide sequence NM_(—)000142); NP_(—)001156685 (correspondingnucleotide sequence NM_(—)001163213); NP_(—)075254 (correspondingnucleotide sequence NM_(—)022965).

SEQ ID NO: 90 is the FGFR3 Amino Acid Sequence, Transcript Variant 1(NP_(—)000133; 806 aa). The location of exons are marked by alternatingunderlining. Amino acids encoded by nucleotides spanning exons areshaded in gray.

SEQ ID NO: 91 is the FGFR3 Nucleotide Sequence, Transcript Variant 1(NM_(—)000142; 4304 bp).

   1 gtcgcgggca gctggcgccg cgcggtcctg ctctgccggt cgcacggacg caccggcggg  61 ccgccggccg gagggacggg gcgggagctg ggcccgcgga cagcgagccg gagcgggagc 121 cgcgcgtagc gagccgggct ccggcgctcg ccagtctccc gagcggcgcc cgcctcccgc 181 cggtgcccgc gccgggccgt ggggggcagc atgcccgcgc gcgctgcctg aggacgccgc 241 ggcccccgcc cccgccatgg gcgcccctgc ctgcgccctc gcgctctgcg tggccgtggc 301 catcgtggcc ggcgcctcct cggagtcctt ggggacggag cagcgcgtcg tggggcgagc 361 ggcagaagtc ccgggcccag agcccggcca gcaggagcag ttggtcttcg gcagcgggga 421 tgctgtggag ctgagctgtc ccccgcccgg gggtggtccc atggggccca ctgtctgggt 481 caaggatggc acagggctgg tgccctcgga gcgtgtcctg gtggggcccc agcggctgca 541 ggtgctgaat gcctcccacg aggactccgg ggcctacagc tgccggcagc ggctcacgca 601 gcgcgtactg tgccacttca gtgtgcgggt gacagacgct ccatcctcgg gagatgacga 661 agacggggag gacgaggctg aggacacagg tgtggacaca ggggcccctt actggacacg 721 gcccgagcgg atggacaaga agctgctggc cgtgccggcc gccaacaccg tccgcttccg 781 ctgcccagcc gctggcaacc ccactccctc catctcctgg ctgaagaacg gcagggagtt 841 ccgcggcgag caccgcattg gaggcatcaa gctgcggcat cagcagtgga gcctggtcat 901 ggaaagcgtg gtgccctcgg accgcggcaa ctacacctgc gtcgtggaga acaagtttgg 961 cagcatccgg cagacgtaca cgctggacgt gctggagcgc tccccgcacc ggcccatcct1021 gcaggcgggg ctgccggcca accagacggc ggtgctgggc agcgacgtgg agttccactg1081 caaggtgtac agtgacgcac agccccacat ccagtggctc aagcacgtgg aggtgaatgg1141 cagcaaggtg ggcccggacg gcacacccta cgttaccgtg ctcaagacgg cgggcgctaa1201 caccaccgac aaggagctag aggttctctc cttgcacaac gtcacctttg aggacgccgg1261 ggagtacacc tgcctggcgg gcaattctat tgggttttct catcactctg cgtggctggt1321 ggtgctgcca gccgaggagg agctggtgga ggctgacgag gcgggcagtg tgtatgcagg1381 catcctcagc tacggggtgg gcttcttcct gttcatcctg gtggtggcgg ctgtgacgct1441 ctgccgcctg cgcagccccc ccaagaaagg cctgggctcc cccaccgtgc acaagatctc1501 ccgcttcccg ctcaagcgac aggtgtccct ggagtccaac gcgtccatga gctccaacac1561 accactggtg cgcatcgcaa ggctgtcctc aggggagggc cccacgctgg ccaatgtctc1621 cgagctcgag ctgcctgccg accccaaatg ggagctgtct cgggcccggc tgaccctggg1681 caagcccctt ggggagggct gcttcggcca ggtggtcatg gcggaggcca tcggcattga1741 caaggaccgg gccgccaagc ctgtcaccgt agccgtgaag atgctgaaag acgatgccac1801 tgacaaggac ctgtcggacc tggtgtctga gatggagatg atgaagatga tcgggaaaca1861 caaaaacatc atcaacctgc tgggcgcctg cacgcagggc gggcccctgt acgtgctggt1921 ggagtacgcg gccaagggta acctgcggga gtttctgcgg gcgcggcggc ccccgggcct1981 ggactactcc ttcgacacct gcaagccgcc cgaggagcag ctcaccttca aggacctggt2041 gtcctgtgcc taccaggtgg cccggggcat ggagtacttg gcctcccaga agtgcatcca2101 cagggacctg gctgcccgca atgtgctggt gaccgaggac aacgtgatga agatcgcaga2161 cttcgggctg gcccgggacg tgcacaacct cgactactac aagaagacaa ccaacggccg2221 gctgcccgtg aagtggatgg cgcctgaggc cttgtttgac cgagtctaca ctcaccagag2281 tgacgtctgg tcctttgggg tcctgctctg ggagatcttc acgctggggg gctccccgta2341 ccccggcatc cctgtggagg agctcttcaa gctgctgaag gagggccacc gcatggacaa2401 gcccgccaac tgcacacacg acctgtacat gatcatgcgg gagtgctggc atgccgcgcc2461 ctcccagagg cccaccttca agcagctggt ggaggacctg gaccgtgtcc ttaccgtgac2521 gtccaccgac gagtacctgg acctgtcggc gcctttcgag cagtactccc cgggtggcca2581 ggacaccccc agctccagct cctcagggga cgactccgtg tttgcccacg acctgctgcc2641 cccggcccca cccagcagtg ggggctcgcg gacgtgaagg gccactggtc cccaacaatg2701 tgaggggtcc ctagcagccc accctgctgc tggtgcacag ccactccccg gcatgagact2761 cagtgcagat ggagagacag ctacacagag ctttggtctg tgtgtgtgtg tgtgcgtgtg2821 tgtgtgtgtg tgtgcacatc cgcgtgtgcc tgtgtgcgtg cgcatcttgc ctccaggtgc2881 agaggtaccc tgggtgtccc cgctgctgtg caacggtctc ctgactggtg ctgcagcacc2941 gaggggcctt tgttctgggg ggacccagtg cagaatgtaa gtgggcccac ccggtgggac3001 ccccgtgggg cagggagctg ggcccgacat ggctccggcc tctgcctttg caccacggga3061 catcacaggg tgggcctcgg cccctcccac acccaaagct gagcctgcag ggaagcccca3121 catgtccagc accttgtgcc tggggtgtta gtggcaccgc ctccccacct ccaggctttc3181 ccacttccca ccctgcccct cagagactga aattacgggt acctgaagat gggagccttt3241 accttttatg caaaaggttt attccggaaa ctagtgtaca tttctataaa tagatgctgt3301 gtatatggta tatatacata tatatatata acatatatgg aagaggaaaa ggctggtaca3361 acggaggcct gcgaccctgg gggcacagga ggcaggcatg gccctgggcg gggcgtgggg3421 gggcgtggag ggaggcccca gggggtctca cccatgcaag cagaggacca gggccttttc3481 tggcaccgca gttttgtttt aaaactggac ctgtatattt gtaaagctat ttatgggccc3541 ctggcactct tgttcccaca ccccaacact tccagcattt agctggccac atggcggaga3601 gttttaattt ttaacttatt gacaaccgag aaggtttatc ccgccgatag agggacggcc3661 aagaatgtac gtccagcctg ccccggagct ggaggatccc ctccaagcct aaaaggttgt3721 taatagttgg aggtgattcc agtgaagata ttttatttcc tttgtccttt ttcaggagaa3781 ttagatttct ataggatttt tctttaggag atttattttt tggacttcaa agcaagctgg3841 tattttcata caaattcttc taattgctgt gtgtcccagg cagggagacg gtttccaggg3901 aggggccggc cctgtgtgca ggttccgatg ttattagatg ttacaagttt atatatatct3961 atatatataa tttattgagt ttttacaaga tgtatttgtt gtagacttaa cacttcttac4021 gcaatgcttc tagagtttta tagcctggac tgctaccttt caaagcttgg agggaagccg4081 tgaattcagt tggttcgttc tgtactgtta ctgggccctg agtctgggca gctgtccctt4141 gcttgcctgc agggccatgg ctcagggtgg tctcttcttg gggcccagtg catggtggcc4201 agaggtgtca cccaaaccgg caggtgcgat tttgttaacc cagcgacgaa ctttccgaaa4261 aataaagaca cctggttgct aacctggaaa aaaaaaaaaa aaaa

SEQ ID NO: 528 is the FGFR3 wt cDNA Nucleotide Sequence corresponding tothe coding sequence of FGFR3 (2421 bp) (NM_(—)000142.4 NP_(—)000133.1).The location of exons are marked by alternating underlining.

ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGTCCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGAGCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGGCCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGCTGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGTACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAGGCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGCTGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTCCTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAGTGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGTTTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACACCCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCACAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCACTCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATGCAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAGCGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGTCCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCTGTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAGGCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAACATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAGGGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGCCGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTACTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTGATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACGGCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTGGAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGTACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGACCTGGACCGTGTCCTTACCGTGACGTCCACCGACGAGTACCTGGACCTGTCGGCGCCTTTCGAGCAGTACTCCCCGGGTGGCCAGGACACCCCCAGCTCCAGCTCCTCAGGGGACGACTCCGTGTTTGCCCACGACCTGCTGCCCCCGGCCCCACCCAGCAGTGGGGGCTCGCGGACGTGA

The Genbank ID for the TACC3 gene is 10460. SEQ ID NO: 92 is the TACC3Amino Acid Sequence (NP_(—)006333) (838 aa). The location of exons aremarked by alternating underlining. Amino acids encoded by nucleotidesspanning exons are shaded in gray. Double underlining indicates theamino acid encoded by the nucleotides shaded in grey in SEQ ID NO: 529.

SEQ ID NO: 93 is the TACC3 Nucleotide Sequence (NM_(—)006342) (2847 bp):

   1 gcgtttgaaa ctccggcgcg ccggcggcca tcaagggcta gaagcgcgac ggcggtagca  61 gctaggcttg gcccccggcg tggagcagac gcggacccct ccttcctggc ggcggcggcg 121 cgggctcaga gcccggcaac gggcgggcgg gcagaatgag tctgcaggtc ttaaacgaca 181 aaaatgtcag caatgaaaaa aatacagaaa attgcgactt cctgttttcg ccaccagaag 241 ttaccggaag atcgtctgtt cttcgtgtgt cacagaaaga aaatgtgcca cccaagaacc 301 tggccaaagc tatgaaggtg acttttcaga cacctctgcg ggatccacag acgcacagga 361 ttctaagtcc tagcatggcc agcaaacttg aggctccttt cactcaggat gacacccttg 421 gactggaaaa ctcacacccg gtctggacac agaaagagaa ccaacagctc atcaaggaag 481 tggatgccaa aactactcat ggaattctac agaaaccagt ggaggctgac accgacctcc 541 tgggggatgc aagcccagcc tttgggagtg gcagctccag cgagtctggc ccaggtgccc 601 tggctgacct ggactgctca agctcttccc agagcccagg aagttctgag aaccaaatgg 661 tgtctccagg aaaagtgtct ggcagccctg agcaagccgt ggaggaaaac cttagttcct 721 attccttaga cagaagagtg acacccgcct ctgagaccct agaagaccct tgcaggacag 781 agtcccagca caaagcggag actccgcacg gagccgagga agaatgcaaa gcggagactc 841 cgcacggagc cgaggaggaa tgccggcacg gtggggtctg tgctcccgca gcagtggcca 901 cttcgcctcc tggtgcaatc cctaaggaag cctgcggagg agcacccctg cagggtctgc 961 ctggcgaagc cctgggctgc cctgcgggtg tgggcacccc cgtgccagca gatggcactc1021 agacccttac ctgtgcacac acctctgctc ctgagagcac agccccaacc aaccacctgg1081 tggctggcag ggccatgacc ctgagtcctc aggaagaagt ggctgcaggc caaatggcca1141 gctcctcgag gagcggacct gtaaaactag aatttgatgt atctgatggc gccaccagca1201 aaagggcacc cccaccaagg agactgggag agaggtccgg cctcaagcct cccttgagga1261 aagcagcagt gaggcagcaa aaggccccgc aggaggtgga ggaggacgac ggtaggagcg1321 gagcaggaga ggaccccccc atgccagctt ctcggggctc ttaccacctc gactgggaca1381 aaatggatga cccaaacttc atcccgttcg gaggtgacac caagtctggt tgcagtgagg1441 cccagccccc agaaagccct gagaccaggc tgggccagcc agcggctgaa cagttgcatg1501 ctgggcctgc cacggaggag ccaggtccct gtctgagcca gcagctgcat tcagcctcag1561 cggaggacac gcctgtggtg cagttggcag ccgagacccc aacagcagag agcaaggaga1621 gagccttgaa ctctgccagc acctcgcttc ccacaagctg tccaggcagt gagccagtgc1681 ccacccatca gcaggggcag cctgccttgg agctgaaaga ggagagcttc agagaccccg1741 ctgaggttct aggcacgggc gcggaggtgg attacctgga gcagtttgga acttcctcgt1801 ttaaggagtc ggccttgagg aagcagtcct tatacctcaa gttcgacccc ctcctgaggg1861 acagtcctgg tagaccagtg cccgtggcca ccgagaccag cagcatgcac ggtgcaaatg1921 agactccctc aggacgtccg cgggaagcca agcttgtgga gttcgatttc ttgggagcac1981 tggacattcc tgtgccaggc ccacccccag gtgttcccgc gcctgggggc ccacccctgt2041 ccaccggacc tatagtggac ctgctccagt acagccagaa ggacctggat gcagtggtaa2101 aggcgacaca ggaggagaac cgggagctga ggagcaggtg tgaggagctc cacgggaaga2161 acctggaact ggggaagatc atggacaggt tcgaagaggt tgtgtaccag gccatggagg2221 aagttcagaa gcagaaggaa ctttccaaag ctgaaatcca gaaagttcta aaagaaaaag2281 accaacttac cacagatctg aactccatgg agaagtcctt ctccgacctc ttcaagcgtt2341 ttgagaaaca gaaagaggtg atcgagggct accgcaagaa cgaagagtca ctgaagaagt2401 gcgtggagga ttacctggca aggatcaccc aggagggcca gaggtaccaa gccctgaagg2461 cccacgcgga ggagaagctg cagctggcaa acgaggagat cgcccaggtc cggagcaagg2521 cccaggcgga agcgttggcc ctccaggcca gcctgaggaa ggagcagatg cgcatccagt2581 cgctggagaa gacagtggag cagaagacta aagagaacga ggagctgacc aggatctgcg2641 acgacctcat ctccaagatg gagaagatct gacctccacg gagccgctgt ccccgccccc2701 ctgctcccgt ctgtctgtcc tgtctgattc tcttaggtgt catgttcttt tttctgtctt2761 gtcttcaact tttttaaaaa ctagattgct ttgaaaacat gactcaataa aagtttcctt2821 tcaatttaaa cactgaaaaa aaaaaaa

SEQ ID NO: 529 is the TACC3 wt cDNA Nucleotide Sequence corresponding tothe coding sequence of TACC3 (2517 bp) (NM_(—)006342.2, NP_(—)006333.1).The location of exons are marked by alternating underlining.

ATGAGTCTGCAGGTCTTAAACGACAAAAATGTCAGCAATGAAAAAAATACAGAAAATTGCGACTTCCTGTTTTCGCCACCAGAAGTTACCGGAAGATCGTCTGTTCTTCGTGTGTCACAGAAAGAAAATGTGCCACCCAAGAACCTGGCCAAAGCTATGAAGGTGACTTTTCAGACACCTCTGCGGGATCCACAGACGCACAGGATTCTAAGTCCTAGCATGGCCAGCAAACTTGAGGCTCCTTTCACTCAGGATGACACCCTTGGACTGGAAAACTCAC

TCTACAGAAACCAGTGGAGGCTGACACCGACCTCCTGGGGGATGCAAGCCCAGCCTTTGGGAGTGGCAGCTCCAGCGAGTCTGGCCCAGGTGCCCTGGCTGACCTGGACTGCTCAAGCTCTTCCCAGAGCCCAGGAAGTTCTGAGAACCAAATGGTGTCTCCAGGAAAAGTGTCTGGCAGCCCTGAGCAAGCCGTGGAGGAAAACCTTAGTTCCTATTCCTTAGACAGAAGAGTGACACCCGCCTCTGAGACCCTAGAAGACCCTTGCAGGACAGAGTCCCAGCACAAAGCGGAGACTCCGCACGGAGCCGAGGAAGAATGCAAAGCGGAGACTCCGCACGGAGCCGAGGAGGAATGCCGGCACGGTGGGGTCTGTGCTCCCGCAGCAGTGGCCACTTCGCCTCCTGGTGCAATCCCTAAGGAAGCCTGCGGAGGAGCACCCCTGCAGGGTCTGCCTGGCGAAGCCCTGGGCTGCCCTGCGGGTGTGGGCACCCCCGTGCCAGCAGATGGCACTCAGACCCTTACCTGTGCACACACCTCTGCTCCTGAGAGCACAGCCCCAACCAACCACCTGGTGGCTGGCAGGGCCATGACCCTGAGTCCTCAGGAAGAAGTGGCTGCAGGCCAAATGGCCAGCTCCTCGAGGAGCGGACCTGTAAAACTAGAATTTGATGTATCTGATGGCGCCACCAGCAAAAGGGCACCCCCACCAAGGAGACTGGGAGAGAGGTCCGGCCTCAAGCCTCCCTTGAGGAAAGCAGCAGTGAGGCAGCAAAAGGCCCCGCAGGAGGTGGAGGAGGACGACGGTAGGAGCGGAGCAGGAGAGGACCCCCCCATGCCAGCTTCTCGGGGCTCTTACCACCTCGACTGGGACAAAATGGATGACCCAAACTTCATCCCGTTCGGAGGTGACACCAAGTCTGGTTGCAGTGAGGCCCAGCCCCCAGAAAGCCCTGAGACCAGGCTGGGCCAGCCAGCGGCTGAACAGTTGCATGCTGGGCCTGCCACGGAGGAGCCAGGTCCCTGTCTGAGCCAGCAGCTGCATTCAGCCTCAGCGGAGGACACGCCTGTGGTGCAGTTGGCAGCCGAGACCCCAACAGCAGAGAGCAAGGAGAGAGCCTTGAACTCTGCCAGCACCTCGCTTCCCACAAGCTGTCCAGGCAGTGAGCCAGTGCCCACCCATCAGCAGGGGCAGCCTGCCTTGGAGCTGAAAGAGGAGAGCTTCAGAGACCCCGCTGAGGTTCTAGGCACGGGCGCGGAGGTGGATTACCTGGAGCAGTTTGGAACTTCCTCGTTTAAGGAGTCGGCCTTGAGGAAGCAGTCCTTATACCTCAAGTTCGACCCCCTCCTGAGGGACAGTCCTGGTAGACCAGTGCCCGTGGCCACCGAGACCAGCAGCATGCACGGTGCAAATGAGACTCCCTCAGGACGTCCGCGGGAAGCCAAGCTTGTGGAGTTCGATTTCTTGGGAGCACTGGACATTCCTGTGCCAGGCCCACCCCCAGGTGTTCCCGCGCCTGGGGGCCCACCCCTGTCCACCGGACCTATAGTGGACCTGCTCCAGTACAGCCAGAAGGACCTGGATGCAGTGGTAAAGGCGACACAGGAGGAGAACCGGGAGCTGAGGAGCAGGTGTGAGGAGCTCCACGGGAAGAACCTGGAACTGGGGAAGATCATGGACAGGTTCGAAGAGGTTGTGTACCAGGCCATGGAGGAAGTTCAGAAGCAGAAGGAACTTTCCAAAGCTGAAATCCAGAAAGTTCTAAAAGAAAAAGACCAACTTACCACAGATCTGAACTCCATGGAGAAGTCCTTCTCCGACCTCTTCAAGCGTTTTGAGAAACAGAAAGAGGTGATCGAGGGCTACCGCAAGAACGAAGAGTCACTGAAGAAGTGCGTGGAGGATTACCTGGCAAGGATCACCCAGGAGGGCCAGAGGTACCAAGCCCTGAAGGCCCACGCGGAGGAGAAGCTGCAGCTGGCAAACGAGGAGATCGCCCAGGTCCGGAGCAAGGCCCAGGCGGAAGCGTTGGCCCTCCAGGCCAGCCTGAGGAAGGAGCAGATGCGCATCCAGTCGCTGGAGAAGACAGTGGAGCAGAAGACTAAAGAGAACGAGGAGCTGACCAGGATCTGCGACGACCTCATCTCCAAGATGGAGAAGATCTGA

SEQ ID NO: 94 is the nucleotide sequence of FGFR3-TACC3.

   1 gtcgcgggca gctggcgccg cgcggtcctg ctctgccggt cgcacggacg caccggcggg  61 ccgccggccg gagggacggg gcgggagctg ggcccgcgga cagcgagccg gagcgggagc 121 cgcgcgtagc gagccgggct ccggcgctcg ccagtctccc gagcggcgcc cgcctcccgc 181 cggtgcccgc gccgggccgt ggggggcagc atgcccgcgc gcgctgcctg aggacgccgc 241 ggcccccgcc cccgccatgg gcgcccctgc ctgcgccctc gcgctctgcg tggccgtggc 301 catcgtggcc ggcgcctcct cggagtcctt ggggacggag cagcgcgtcg tggggcgagc 361 ggcagaagtc ccgggcccag agcccggcca gcaggagcag ttggtcttcg gcagcgggga 421 tgctgtggag ctgagctgtc ccccgcccgg gggtggtccc atggggccca ctgtctgggt 481 caaggatggc acagggctgg tgccctcgga gcgtgtcctg gtggggcccc agcggctgca 541 ggtgctgaat gcctcccacg aggactccgg ggcctacagc tgccggcagc ggctcacgca 601 gcgcgtactg tgccacttca gtgtgcgggt gacagacgct ccatcctcgg gagatgacga 661 agacggggag gacgaggctg aggacacagg tgtggacaca ggggcccctt actggacacg 721 gcccgagcgg atggacaaga agctgctggc cgtgccggcc gccaacaccg tccgcttccg 781 ctgcccagcc gctggcaacc ccactccctc catctcctgg ctgaagaacg gcagggagtt 841 ccgcggcgag caccgcattg gaggcatcaa gctgcggcat cagcagtgga gcctggtcat 901 ggaaagcgtg gtgccctcgg accgcggcaa ctacacctgc gtcgtggaga acaagtttgg 961 cagcatccgg cagacgtaca cgctggacgt gctggagcgc tccccgcacc ggcccatcct1021 gcaggcgggg ctgccggcca accagacggc ggtgctgggc agcgacgtgg agttccactg1081 caaggtgtac agtgacgcac agccccacat ccagtggctc aagcacgtgg aggtgaatgg1141 cagcaaggtg ggcccggacg gcacacccta cgttaccgtg ctcaagacgg cgggcgctaa1201 caccaccgac aaggagctag aggttctctc cttgcacaac gtcacctttg aggacgccgg1261 ggagtacacc tgcctggcgg gcaattctat tgggttttct catcactctg cgtggctggt1321 ggtgctgcca gccgaggagg agctggtgga ggctgacgag gcgggcagtg tgtatgcagg1381 catcctcagc tacggggtgg gcttcttcct gttcatcctg gtggtggcgg ctgtgacgct1441 ctgccgcctg cgcagccccc ccaagaaagg cctgggctcc cccaccgtgc acaagatctc1501 ccgcttcccg ctcaagcgac aggtgtccct ggagtccaac gcgtccatga gctccaacac1561 accactggtg cgcatcgcaa ggctgtcctc aggggagggc cccacgctgg ccaatgtctc1621 cgagctcgag ctgcctgccg accccaaatg ggagctgtct cgggcccggc tgaccctggg1681 caagcccctt ggggagggct gcttcggcca ggtggtcatg gcggaggcca tcggcattga1741 caaggaccgg gccgccaagc ctgtcaccgt agccgtgaag atgctgaaag acgatgccac1801 tgacaaggac ctgtcggacc tggtgtctga gatggagatg atgaagatga tcgggaaaca1861 caaaaacatc atcaacctgc tgggcgcctg cacgcagggc gggcccctgt acgtgctggt1921 ggagtacgcg gccaagggta acctgcggga gtttctgcgg gcgcggcggc ccccgggcct1981 ggactactcc ttcgacacct gcaagccgcc cgaggagcag ctcaccttca aggacctggt2041 gtcctgtgcc taccaggtgg cccggggcat ggagtacttg gcctcccaga agtgcatcca2101 cagggacctg gctgcccgca atgtgctggt gaccgaggac aacgtgatga agatcgcaga2161 cttcgggctg gcccgggacg tgcacaacct cgactactac aagaagacaa ccaacggccg2221 gctgcccgtg aagtggatgg cgcctgaggc cttgtttgac cgagtctaca ctcaccagag2281 tgacgtctgg tcctttgggg tcctgctctg ggagatcttc acgctggggg gctccccgta2341 ccccggcatc cctgtggagg agctcttcaa gctgctgaag gagggccacc gcatggacaa2401 gcccgccaac tgcacacacg acctgtacat gatcatgcgg gagtgctggc atgccgcgcc2461 ctcccagagg cccaccttca agcagctggt ggaggacctg gaccgtgtcc ttaccgtgac2521 gtccaccgac tttaaggagt cggccttgag gaagcagtcc ttatacctca agttcgaccc2581 cctcctgagg gacagtcctg gtagaccagt gcccgtggcc accgagacca gcagcatgca2641 cggtgcaaat gagactccct caggacgtcc gcgggaagcc aagcttgtgg agttcgattt2701 cttgggagca ctggacattc ctgtgccagg cccaccccca ggtgttcccg cgcctggggg2761 cccacccctg tccaccggac ctatagtgga cctgctccag tacagccaga aggacctgga2821 tgcagtggta aaggcgacac aggaggagaa ccgggagctg aggagcaggt gtgaggagct2881 ccacgggaag aacctggaac tggggaagat catggacagg ttcgaagagg ttgtgtacca2941 ggccatggag gaagttcaga agcagaagga actttccaaa gctgaaatcc agaaagttct3001 aaaagaaaaa gaccaactta ccacagatct gaactccatg gagaagtcct tctccgacct3061 cttcaagcgt tttgagaaac agaaagaggt gatcgagggc taccgcaaga acgaagagtc3121 actgaagaag tgcgtggagg attacctggc aaggatcacc caggagggcc agaggtacca3181 agccctgaag gcccacgcgg aggagaagct gcagctggca aacgaggaga tcgcccaggt3241 ccggagcaag gcccaggcgg aagcgttggc cctccaggcc agcctgagga aggagcagat3301 gcgcatccag tcgctggaga agacagtgga gcagaagact aaagagaacg aggagctgac3361 caggatctgc gacgacctca tctccaagat ggagaagatc tgacctccac ggagccgctg3421 tccccgcccc cctgctcccg tctgtctgtc ctgtctgatt ctcttaggtg tcatgttctt3481 ttttctgtct tgtcttcaac ttttttaaaa actagattgc tttgaaaaca tgactcaata3541 aaagtttcct ttcaatttaa acactgaaaa aaaaaaaa

SEQ ID NO: 530 is the nucleotide sequence (cDNA) of FGFR3ex17-TACC3ex11.The sequence corresponding to FGFR3 is underlined. The sequencecorresponding to TACC3 is shaded:

ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGTCCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGAGCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGGCCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGCTGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGTACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAGGCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGCTGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTCCTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAGTGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGTTTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACACCCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCACAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCACTCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATGCAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAGCGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGTCCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCTGTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAGGCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAACATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAGGGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGCCGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTACTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTGATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACGGCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTGGAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGTACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGA

SEQ ID NO: 531 is the nucleotide sequence (cDNA) of FGFR3ex17-TACC3ex8.The sequence corresponding to FGFR3 is underlined. The sequencecorresponding to TACC3 is shaded:

ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGTCCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGAGCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGGCCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGCTGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGTACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAGGCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGCTGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTCCTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAGTGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGTTTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACACCCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCACAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCACTCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATGCAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAGCGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGTCCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCTGTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAGGCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAACATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAGGGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGCCGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTACTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTGATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACGGCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTGGAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGTACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGA

SEQ ID NO: 532 is the nucleotide sequence (cDNA) of FGFR3ex17-TACC3ex10.The sequence corresponding to FGFR3 is underlined. The sequencecorresponding to TACC3 is shaded:

ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGTCCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGAGCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGGCCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGCTGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGTACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAGGCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGCTGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTCCTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAGTGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGTTTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACACCCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCACAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCACTCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATGCAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAGCGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGTCCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCTGTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAGGCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAACATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAGGGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGCCGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTACTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTGATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACGGCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTGGAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGTACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGA

SEQ ID NO: 533 is the nucleotide sequence (cDNA) of FGFR3ex17-TACC3ex6.The sequence corresponding to FGFR3 is underlined. The sequencecorresponding to TACC3 is shaded:

ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGTCCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGAGCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGGCCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGCTGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGTACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAGGCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGCTGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTCCTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAGTGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGTTTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACACCCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCACAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCACTCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATGCAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAGCGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGTCCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCTGTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAGGCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAACATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAGGGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGCCGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTACTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTGATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACGGCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTGGAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGTACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGA

SEQ ID NO: 534 is the nucleotide sequence (cDNA) of FGFR3ex18-TACC3ex13.The sequence corresponding to FGFR3 is underlined. The sequencecorresponding to TACC3 is shaded:

ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGTCCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGAGCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGGCCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGCTGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGTACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAGGCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGCTGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTCCTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAGTGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGTTTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACACCCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCACAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCACTCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATGCAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAGCGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGTCCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCTGTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAGGCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAACATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAGGGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGCCGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTACTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTGATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACGGCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTGGAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGTACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGACCTGGACCGTGTCCTTACCGTGACGTCCACCGACGAGTACCTGGACCTGTCGGCGCCTTTCGAGCAGTAC

SEQ ID NO: 535 is the nucleotide sequence (cDNA) ofFGFR3ex18-TACC3ex9_INS66BP. The sequence corresponding to FGFR3 isunderlined. The sequence corresponding to TACC3 is shaded. The sequencecorresponding the the 66 bp intronic insert is double underlined:

ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGTCCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGAGCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGGCCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGCTGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGTACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAGGCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGCTGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTCCTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAGTGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGTTTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACACCCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCACAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCACTCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATGCAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAGCGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGTCCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCTGTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAGGCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAACATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAGGGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGCCGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTACTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTGATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACGGCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTGGAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGTACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGACCTGGACCGTGTCCTTACCGTGACGTCCACCGACGAGTACCTGGACCTGTCGGCGC AGGAGCAACGGCAG

SEQ ID NO: 536 is the nucleotide sequence (cDNA) of FGFR3ex18-TACC3ex5.The sequence corresponding to FGFR3 is underlined. The sequencecorresponding to TACC3 is shaded:

ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGTCCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGAGCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGGCCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGCTGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGTACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAGGCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGCTGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTCCTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAGTGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGTTTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACACCCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCACAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCACTCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATGCAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAGCGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGTCCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCTGTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAGGCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAACATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAGGGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGCCGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTACTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTGATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACGGCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTGGAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGTACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGA

SEQ ID NO: 537 is the nucleotide sequence (cDNA) ofFGFR3ex18-TACC3ex5_INS33 bp. The sequence corresponding to FGFR3 isunderlined. The sequence corresponding to TACC3 is shaded. The sequencecorresponding the the 33 bp intronic insert is double underlined:

ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGTCCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGAGCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGGCCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGCTGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGTACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAGGCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGCTGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTCCTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAGTGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGTTTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACACCCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCACAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCACTCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATGCAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAGCGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGTCCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCTGTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAGGCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAACATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAGGGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGCCGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTACTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTGATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACGGCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTGGAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGTACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGACCTGGACCGTGTCCTTACCGTGACGTCCACCGACGAGTACCTGGACCTGTCGGCGCCTTTCGAGCAGTACTCCCCGGGTGGCCAGGACACCCCCAGCTCCAGCTCCTCAGGGGAC GTGCGTGAGCCACCGCACCCGGCGT

SEQ ID NO: 538 is the nucleotide sequence (cDNA) of FGFR3ex18-TACC3ex4.The sequence corresponding to FGFR3 is underlined. The sequencecorresponding to TACC3 is shaded.

ATGGGCGCCCCTGCCTGCGCCCTCGCGCTCTGCGTGGCCGTGGCCATCGTGGCCGGCGCCTCCTCGGAGTCCTTGGGGACGGAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGAGCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGGGCCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGCTGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGTACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAGGCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGCTGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTCCTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCAGTGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGTTTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACACCCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCACAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCACTCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATGCAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAGCGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGTCCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCTGTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGAGGCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAACATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAGGGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGCCGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTACTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGTGATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACGGCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTGGAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGTACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGACCTGGACCGTGTCCTTACCGTGACGTCCACCGACGAGTACCTGGACCTGTCGGCGCCTTTCGAGCAGTAC

The Genbank ID for the FGFR1 gene is 2260. Eight isoforms are listed forFGFR1, e.g., having Genebank Accession Nos. NP_(—)001167534(corresponding nucleotide sequence NM_(—)001174063); NP_(—)001167535(corresponding nucleotide sequence NM_(—)001174064); NP_(—)001167536(corresponding nucleotide sequence NM_(—)001174065); NP_(—)001167537(corresponding nucleotide sequence NM_(—)001174066); NP_(—)001167538(corresponding nucleotide sequence NM_(—)001174067); NP_(—)056934(corresponding nucleotide sequence NM_(—)015850); NP_(—)075593(corresponding nucleotide sequence NM_(—)023105); NP_(—)075594(corresponding nucleotide sequence NM_(—)023106); NP_(—)075598(corresponding nucleotide sequence NM_(—)023110).

SEQ ID NO: 146 is the FGFR1 Amino Acid Sequence for isoform 10, havingGenebank Accession No. NP_(—)001167534 (820 aa):

   1 MWSWKCLLFW AVLVTATLCT ARPSPTLPEQ AQPWGAPVEV ESFLVHPGDL LQLRCRLRDD  61 VQSINWLRDG VQLAESNRTR ITGEEVEVQD SVPADSGLYA CVTSSPSGSD TTYFSVNVSD 121 ALPSSEDDDD DDDSSSEEKE TDNTKPNRMP VAPYWTSPEK MEKKLHAVPA AKTVKFKCPS 181 SGTPNPTLRW LKNGKEFKPD HRIGGYKVRY ATWSIIMDSV VPSDKGNYTC IVENEYGSIN 241 HTYQLDVVER SPHRPILQAG LPANKTVALG SNVEFMCKVY SDPQPHIQWL KHIEVNGSKI 301 GPDNLPYVQI LKTAGVNTTD KEMEVLHLRN VSFEDAGEYT CLAGNSIGLS HHSAWLTVLE 361 ALEERPAVMT SPLYLEIIIY CTGAFLISCM VGSVIVYKMK SGTKKSDFHS QMAVHKLAKS 421 IPLRRQVSAD SSASMNSGVL LVRPSRLSSS GTPMLAGVSE YELPEDPRWE LPRDRLVLGK 481 PLGEGCFGQV VLAEAIGLDK DKPNRVTKVA VKMLKSDATE KDLSDLISEM EMMKMIGKHK 541 NIINLLGACT QDGPLYVIVE YASKGNLREY LQARRPPGLE YCYNPSHNPE EQLSSKDLVS 601 CAYQVARGME YLASKKCIHR DLAARNVLVT EDNVMKIADF GLARDIHHID YYKKTTNGRL 661 PVKWMAPEAL FDRIYTHQSD VWSFGVLLWE IFTLGGSPYP GVPVEELFKL LKEGHRMDKP 721 SNCTNELYMM MRDCWHAVPS QRPTFKQLVE DLDRIVALTS NQEYLDLSMP LDQYSPSFPD 781 TRSSTCSSGE DSVFSHEPLP EEPCLPRHPA QLANGGLKRR

SEQ ID NO: 147 is the FGFR1 Nucleotide Sequence for isoform 10, havingGenebank Accession No. NM_(—)001174063 (5895 bp):

   1 agatgcaggg gcgcaaacgc caaaggagac caggctgtag gaagagaagg gcagagcgcc  61 ggacagctcg gcccgctccc cgtcctttgg ggccgcggct ggggaactac aaggcccagc 121 aggcagctgc agggggcgga ggcggaggag ggaccagcgc gggtgggagt gagagagcga 181 gccctcgcgc cccgccggcg catagcgctc ggagcgctct tgcggccaca ggcgcggcgt 241 cctcggcggc gggcggcagc tagcgggagc cgggacgccg gtgcagccgc agcgcgcgga 301 ggaacccggg tgtgccggga gctgggcggc cacgtccgga cgggaccgag acccctcgta 361 gcgcattgcg gcgacctcgc cttccccggc cgcgagcgcg ccgctgcttg aaaagccgcg 421 gaacccaagg acttttctcc ggtccgagct cggggcgccc cgcagggcgc acggtacccg 481 tgctgcagtc gggcacgccg cggcgccggg gcctccgcag ggcgatggag cccggtctgc 541 aaggaaagtg aggcgccgcc gctgcgttct ggaggagggg ggcacaaggt ctggagaccc 601 cgggtggcgg acgggagccc tccccccgcc ccgcctccgg ggcaccagct ccggctccat 661 tgttcccgcc cgggctggag gcgccgagca ccgagcgccg ccgggagtcg agcgccggcc 721 gcggagctct tgcgaccccg ccaggacccg aacagagccc gggggcggcg ggccggagcc 781 ggggacgcgg gcacacgccc gctcgcacaa gccacggcgg actctcccga ggcggaacct 841 ccacgccgag cgagggtcag tttgaaaagg aggatcgagc tcactgtgga gtatccatgg 901 agatgtggag ccttgtcacc aacctctaac tgcagaactg gg atg tggag ctggaagtgc 961 ctcctcttct gggctgtgct ggtcacagcc acactctgca ccgctaggcc gtccccgacc1021 ttgcctgaac aagcccagcc ctggggagcc cctgtggaag tggagtcctt cctggtccac1081 cccggtgacc tgctgcagct tcgctgtcgg ctgcgggacg atgtgcagag catcaactgg1141 ctgcgggacg gggtgcagct ggcggaaagc aaccgcaccc gcatcacagg ggaggaggtg1201 gaggtgcagg actccgtgcc cgcagactcc ggcctctatg cttgcgtaac cagcagcccc1261 tcgggcagtg acaccaccta cttctccgtc aatgtttcag atgctctccc ctcctcggag1321 gatgatgatg atgatgatga ctcctcttca gaggagaaag aaacagataa caccaaacca1381 aaccgtatgc ccgtagctcc atattggaca tccccagaaa agatggaaaa gaaattgcat1441 gcagtgccgg ctgccaagac agtgaagttc aaatgccctt ccagtgggac cccaaacccc1501 acactgcgct ggttgaaaaa tggcaaagaa ttcaaacctg accacagaat tggaggctac1561 aaggtccgtt atgccacctg gagcatcata atggactctg tggtgccctc tgacaagggc1621 aactacacct gcattgtgga gaatgagtac ggcagcatca accacacata ccagctggat1681 gtcgtggagc ggtcccctca ccggcccatc ctgcaagcag ggttgcccgc caacaaaaca1741 gtggccctgg gtagcaacgt ggagttcatg tgtaaggtgt acagtgaccc gcagccgcac1801 atccagtggc taaagcacat cgaggtgaat gggagcaaga ttggcccaga caacctgcct1861 tatgtccaga tcttgaagac tgctggagtt aataccaccg acaaagagat ggaggtgctt1921 cacttaagaa atgtctcctt tgaggacgca ggggagtata cgtgcttggc gggtaactct1981 atcggactct cccatcactc tgcatggttg accgttctgg aagccctgga agagaggccg2041 gcagtgatga cctcgcccct gtacctggag atcatcatct attgcacagg ggccttcctc2101 atctcctgca tggtggggtc ggtcatcgtc tacaagatga agagtggtac caagaagagt2161 gacttccaca gccagatggc tgtgcacaag ctggccaaga gcatccctct gcgcagacag2221 gtgtctgctg actccagtgc atccatgaac tctggggttc ttctggttcg gccatcacgg2281 ctctcctcca gtgggactcc catgctagca ggggtctctg agtatgagct tcccgaagac2341 cctcgctggg agctgcctcg ggacagactg gtcttaggca aacccctggg agagggctgc2401 tttgggcagg tggtgttggc agaggctatc gggctggaca aggacaaacc caaccgtgtg2461 accaaagtgg ctgtgaagat gttgaagtcg gacgcaacag agaaagactt gtcagacctg2521 atctcagaaa tggagatgat gaagatgatc gggaagcata agaatatcat caacctgctg2581 ggggcctgca cgcaggatgg tcccttgtat gtcatcgtgg agtatgcctc caagggcaac2641 ctgcgggagt acctgcaggc ccggaggccc ccagggctgg aatactgcta caaccccagc2701 cacaacccag aggagcagct ctcctccaag gacctggtgt cctgcgccta ccaggtggcc2761 cgaggcatgg agtatctggc ctccaagaag tgcatacacc gagacctggc agccaggaat2821 gtcctggtga cagaggacaa tgtgatgaag atagcagact ttggcctcgc acgggacatt2881 caccacatcg actactataa aaagacaacc aacggccgac tgcctgtgaa gtggatggca2941 cccgaggcat tatttgaccg gatctacacc caccagagtg atgtgtggtc tttcggggtg3001 ctcctgtggg agatcttcac tctgggcggc tccccatacc ccggtgtgcc tgtggaggaa3061 cttttcaagc tgctgaagga gggtcaccgc atggacaagc ccagtaactg caccaacgag3121 ctgtacatga tgatgcggga ctgctggcat gcagtgccct cacagagacc caccttcaag3181 cagctggtgg aagacctgga ccgcatcgtg gccttgacct ccaaccagga gtacctggac3241 ctgtccatgc ccctggacca gtactccccc agctttcccg acacccggag ctctacgtgc3301 tcctcagggg aggattccgt cttctctcat gagccgctgc ccgaggagcc ctgcctgccc3361 cgacacccag cccagcttgc caatggcgga ctcaaacgcc gctgactgcc acccacacgc3421 cctccccaga ctccaccgtc agctgtaacc ctcacccaca gcccctgctg ggcccaccac3481 ctgtccgtcc ctgtcccctt tcctgctggc aggagccggc tgcctaccag gggccttcct3541 gtgtggcctg ccttcacccc actcagctca cctctccctc cacctcctct ccacctgctg3601 gtgagaggtg caaagaggca gatctttgct gccagccact tcatcccctc ccagatgttg3661 gaccaacacc cctccctgcc accaggcact gcctggaggg cagggagtgg gagccaatga3721 acaggcatgc aagtgagagc ttcctgagct ttctcctgtc ggtttggtct gttttgcctt3781 cacccataag cccctcgcac tctggtggca ggtgccttgt cctcagggct acagcagtag3841 ggaggtcagt gcttcgtgcc tcgattgaag gtgacctctg ccccagatag gtggtgccag3901 tggcttatta attccgatac tagtttgctt tgctgaccaa atgcctggta ccagaggatg3961 gtgaggcgaa ggccaggttg ggggcagtgt tgtggccctg gggcccagcc ccaaactggg4021 ggctctgtat atagctatga agaaaacaca aagtgtataa atctgagtat atatttacat4081 gtctttttaa aagggtcgtt accagagatt tacccatcgg gtaagatgct cctggtggct4141 gggaggcatc agttgctata tattaaaaac aaaaaagaaa aaaaaggaaa atgtttttaa4201 aaaggtcata tattttttgc tacttttgct gttttatttt tttaaattat gttctaaacc4261 tattttcagt ttaggtccct caataaaaat tgctgctgct tcatttatct atgggctgta4321 tgaaaagggt gggaatgtcc actggaaaga agggacaccc acgggccctg gggctaggtc4381 tgtcccgagg gcaccgcatg ctcccggcgc aggttccttg taacctcttc ttcctaggtc4441 ctgcacccag acctcacgac gcacctcctg cctctccgct gcttttggaa agtcagaaaa4501 agaagatgtc tgcttcgagg gcaggaaccc catccatgca gtagaggcgc tgggcagaga4561 gtcaaggccc agcagccatc gaccatggat ggtttcctcc aaggaaaccg gtggggttgg4621 gctggggagg gggcacctac ctaggaatag ccacggggta gagctacagt gattaagagg4681 aaagcaaggg cgcggttgct cacgcctgta atcccagcac tttgggacac cgaggtgggc4741 agatcacttc aggtcaggag tttgagacca gcctggccaa cttagtgaaa ccccatctct4801 actaaaaatg caaaaattat ccaggcatgg tggcacacgc ctgtaatccc agctccacag4861 gaggctgagg cagaatccct tgaagctggg aggcggaggt tgcagtgagc cgagattgcg4921 ccattgcact ccagcctggg caacagagaa aacaaaaagg aaaacaaatg atgaaggtct4981 gcagaaactg aaacccagac atgtgtctgc cccctctatg tgggcatggt tttgccagtg5041 cttctaagtg caggagaaca tgtcacctga ggctagtttt gcattcaggt ccctggcttc5101 gtttcttgtt ggtatgcctc cccagatcgt ccttcctgta tccatgtgac cagactgtat5161 ttgttgggac tgtcgcagat cttggcttct tacagttctt cctgtccaaa ctccatcctg5221 tccctcagga acggggggaa aattctccga atgtttttgg ttttttggct gcttggaatt5281 tacttctgcc acctgctggt catcactgtc ctcactaagt ggattctggc tcccccgtac5341 ctcatggctc aaactaccac tcctcagtcg ctatattaaa gcttatattt tgctggatta5401 ctgctaaata caaaagaaag ttcaatatgt tttcatttct gtagggaaaa tgggattgct5461 gctttaaatt tctgagctag ggattttttg gcagctgcag tgttggcgac tattgtaaaa5521 ttctctttgt ttctctctgt aaatagcacc tgctaacatt acaatttgta tttatgttta5581 aagaaggcat catttggtga acagaactag gaaatgaatt tttagctctt aaaagcattt5641 gctttgagac cgcacaggag tgtctttcct tgtaaaacag tgatgataat ttctgccttg5701 gccctacctt gaagcaatgt tgtgtgaagg gatgaagaat ctaaaagtct tcataagtcc5761 ttgggagagg tgctagaaaa atataaggca ctatcataat tacagtgatg tccttgctgt5821 tactactcaa atcacccaca aatttcccca aagactgcgc tagctgtcaa ataaaagaca5881 gtgaaattga cctga

SEQ ID NO: 185 is the FGFR1 Amino Acid Sequence for isoform 1, havingGenebank Accession No. NP_(—)075598 (822 aa):

   1 MWSWKCLLFW AVLVTATLCT ARPSPTLPEQ AQPWGAPVEV ESFLVHPGDL LQLRCRLRDD  61 VQSINWLRDG VQLAESNRTR ITGEEVEVQD SVPADSGLYA CVTSSPSGSD TTYFSVNVSD 121 ALPSSEDDDD DDDSSSEEKE TDNTKPNRMP VAPYWTSPEK MEKKLHAVPA AKTVKFKCPS 181 SGTPNPTLRW LKNGKEFKPD HRIGGYKVRY ATWSIIMDSV VPSDKGNYTC IVENEYGSIN 241 HTYQLDVVER SPHRPILQAG LPANKTVALG SNVEFMCKVY SDPQPHIQWL KHIEVNGSKI 301 GPDNLPYVQI LKTAGVNTTD KEMEVLHLRN VSFEDAGEYT CLAGNSIGLS HHSAWLTVLE 361 ALEERPAVMT SPLYLEIIIY CTGAFLISCM VGSVIVYKMK SGTKKSDFHS QMAVHKLAKS 421 IPLRRQVTVS ADSSASMNSG VLLVRPSRLS SSGTPMLAGV SEYELPEDPR WELPRDRLVL 481 GKPLGEGCFG QVVLAEAIGL DKDKPNRVTK VAVKMLKSDA TEKDLSDLIS EMEMMKMIGK 541 HKNIINLLGA CTQDGPLYVI VEYASKGNLR EYLQARRPPG LEYCYNPSHN PEEQLSSKDL 601 VSCAYQVARG MEYLASKKCI HRDLAARNVL VTEDNVMKIA DFGLARDIHH IDYYKKTTNG 661 RLPVKWMAPE ALFDRIYTHQ SDVWSFGVLL WEIFTLGGSP YPGVPVEELF KLLKEGHRMD 721 KPSNCTNELY MMMRDCWHAV PSQRPTFKQL VEDLDRIVAL TSNQEYLDLS MPLDQYSPSF 781 PDTRSSTCSS GEDSVFSHEP LPEEPCLPRH PAQLANGGLK RR

SEQ ID NO: 186 is the FGFR1 Nucleotide Sequence for isoform 1, havingGenebank Accession No. NM_(—)023110 (5917 bp):

   1 agatgcaggg gcgcaaacgc caaaggagac caggctgtag gaagagaagg gcagagcgcc  61 ggacagctcg gcccgctccc cgtcctttgg ggccgcggct ggggaactac aaggcccagc 121 aggcagctgc agggggcgga ggcggaggag ggaccagcgc gggtgggagt gagagagcga 181 gccctcgcgc cccgccggcg catagcgctc ggagcgctct tgcggccaca ggcgcggcgt 241 cctcggcggc gggcggcagc tagcgggagc cgggacgccg gtgcagccgc agcgcgcgga 301 ggaacccggg tgtgccggga gctgggcggc cacgtccgga cgggaccgag acccctcgta 361 gcgcattgcg gcgacctcgc cttccccggc cgcgagcgcg ccgctgcttg aaaagccgcg 421 gaacccaagg acttttctcc ggtccgagct cggggcgccc cgcagggcgc acggtacccg 481 tgctgcagtc gggcacgccg cggcgccggg gcctccgcag ggcgatggag cccggtctgc 541 aaggaaagtg aggcgccgcc gctgcgttct ggaggagggg ggcacaaggt ctggagaccc 601 cgggtggcgg acgggagccc tccccccgcc ccgcctccgg ggcaccagct ccggctccat 661 tgttcccgcc cgggctggag gcgccgagca ccgagcgccg ccgggagtcg agcgccggcc 721 gcggagctct tgcgaccccg ccaggacccg aacagagccc gggggcggcg ggccggagcc 781 ggggacgcgg gcacacgccc gctcgcacaa gccacggcgg actctcccga ggcggaacct 841 ccacgccgag cgagggtcag tttgaaaagg aggatcgagc tcactgtgga gtatccatgg 901 agatgtggag ccttgtcacc aacctctaac tgcagaactg ggatgtggag ctggaagtgc 961 ctcctcttct gggctgtgct ggtcacagcc acactctgca ccgctaggcc gtccccgacc1021 ttgcctgaac aagcccagcc ctggggagcc cctgtggaag tggagtcctt cctggtccac1081 cccggtgacc tgctgcagct tcgctgtcgg ctgcgggacg atgtgcagag catcaactgg1141 ctgcgggacg gggtgcagct ggcggaaagc aaccgcaccc gcatcacagg ggaggaggtg1201 gaggtgcagg actccgtgcc cgcagactcc ggcctctatg cttgcgtaac cagcagcccc1261 tcgggcagtg acaccaccta cttctccgtc aatgtttcag atgctctccc ctcctcggag1321 gatgatgatg atgatgatga ctcctcttca gaggagaaag aaacagataa caccaaacca1381 aaccgtatgc ccgtagctcc atattggaca tccccagaaa agatggaaaa gaaattgcat1441 gcagtgccgg ctgccaagac agtgaagttc aaatgccctt ccagtgggac cccaaacccc1501 acactgcgct ggttgaaaaa tggcaaagaa ttcaaacctg accacagaat tggaggctac1561 aaggtccgtt atgccacctg gagcatcata atggactctg tggtgccctc tgacaagggc1621 aactacacct gcattgtgga gaatgagtac ggcagcatca accacacata ccagctggat1681 gtcgtggagc ggtcccctca ccggcccatc ctgcaagcag ggttgcccgc caacaaaaca1741 gtggccctgg gtagcaacgt ggagttcatg tgtaaggtgt acagtgaccc gcagccgcac1801 atccagtggc taaagcacat cgaggtgaat gggagcaaga ttggcccaga caacctgcct1861 tatgtccaga tcttgaagac tgctggagtt aataccaccg acaaagagat ggaggtgctt1921 cacttaagaa atgtctcctt tgaggacgca ggggagtata cgtgcttggc gggtaactct1981 atcggactct cccatcactc tgcatggttg accgttctgg aagccctgga agagaggccg2041 gcagtgatga cctcgcccct gtacctggag atcatcatct attgcacagg ggccttcctc2101 atctcctgca tggtggggtc ggtcatcgtc tacaagatga agagtggtac caagaagagt2161 gacttccaca gccagatggc tgtgcacaag ctggccaaga gcatccctct gcgcagacag2221 gtaacagtgt ctgctgactc cagtgcatcc atgaactctg gggttcttct ggttcggcca2281 tcacggctct cctccagtgg gactcccatg ctagcagggg tctctgagta tgagcttccc2341 gaagaccctc gctgggagct gcctcgggac agactggtct taggcaaacc cctgggagag2401 ggctgctttg ggcaggtggt gttggcagag gctatcgggc tggacaagga caaacccaac2461 cgtgtgacca aagtggctgt gaagatgttg aagtcggacg caacagagaa agacttgtca2521 gacctgatct cagaaatgga gatgatgaag atgatcggga agcataagaa tatcatcaac2581 ctgctggggg cctgcacgca ggatggtccc ttgtatgtca tcgtggagta tgcctccaag2641 ggcaacctgc gggagtacct gcaggcccgg aggcccccag ggctggaata ctgctacaac2701 cccagccaca acccagagga gcagctctcc tccaaggacc tggtgtcctg cgcctaccag2761 gtggcccgag gcatggagta tctggcctcc aagaagtgca tacaccgaga cctggcagcc2821 aggaatgtcc tggtgacaga ggacaatgtg atgaagatag cagactttgg cctcgcacgg2881 gacattcacc acatcgacta ctataaaaag acaaccaacg gccgactgcc tgtgaagtgg2941 atggcacccg aggcattatt tgaccggatc tacacccacc agagtgatgt gtggtctttc3001 ggggtgctcc tgtgggagat cttcactctg ggcggctccc cataccccgg tgtgcctgtg3061 gaggaacttt tcaagctgct gaaggagggt caccgcatgg acaagcccag taactgcacc3121 aacgagctgt acatgatgat gcgggactgc tggcatgcag tgccctcaca gagacccacc3181 ttcaagcagc tggtggaaga cctggaccgc atcgtggcct tgacctccaa ccaggagtac3241 ctggacctgt ccatgcccct ggaccagtac tcccccagct ttcccgacac ccggagctct3301 acgtgctcct caggggagga ttccgtcttc tctcatgagc cgctgcccga ggagccctgc3361 ctgccccgac acccagccca gcttgccaat ggcggactca aacgccgctg actgccaccc3421 acacgccctc cccagactcc accgtcagct gtaaccctca cccacagccc ctgctgggcc3481 caccacctgt ccgtccctgt cccctttcct gctggcagga gccggctgcc taccaggggc3541 cttcctgtgt ggcctgcctt caccccactc agctcacctc tccctccacc tcctctccac3601 ctgctggtga gaggtgcaaa gaggcagatc tttgctgcca gccacttcat cccctcccag3661 atgttggacc aacacccctc cctgccacca ggcactgcct ggagggcagg gagtgggagc3721 caatgaacag gcatgcaagt gagagcttcc tgagctttct cctgtcggtt tggtctgttt3781 tgccttcacc cataagcccc tcgcactctg gtggcaggtg ccttgtcctc agggctacag3841 cagtagggag gtcagtgctt cgtgcctcga ttgaaggtga cctctgcccc agataggtgg3901 tgccagtggc ttattaattc cgatactagt ttgctttgct gaccaaatgc ctggtaccag3961 aggatggtga ggcgaaggcc aggttggggg cagtgttgtg gccctggggc ccagccccaa4021 actgggggct ctgtatatag ctatgaagaa aacacaaagt gtataaatct gagtatatat4081 ttacatgtct ttttaaaagg gtcgttacca gagatttacc catcgggtaa gatgctcctg4141 gtggctggga ggcatcagtt gctatatatt aaaaacaaaa aagaaaaaaa aggaaaatgt4201 ttttaaaaag gtcatatatt ttttgctact tttgctgttt tattttttta aattatgttc4261 taaacctatt ttcagtttag gtccctcaat aaaaattgct gctgcttcat ttatctatgg4321 gctgtatgaa aagggtggga atgtccactg gaaagaaggg acacccacgg gccctggggc4381 taggtctgtc ccgagggcac cgcatgctcc cggcgcaggt tccttgtaac ctcttcttcc4441 taggtcctgc acccagacct cacgacgcac ctcctgcctc tccgctgctt ttggaaagtc4501 agaaaaagaa gatgtctgct tcgagggcag gaaccccatc catgcagtag aggcgctggg4561 cagagagtca aggcccagca gccatcgacc atggatggtt tcctccaagg aaaccggtgg4621 ggttgggctg gggagggggc acctacctag gaatagccac ggggtagagc tacagtgatt4681 aagaggaaag caagggcgcg gttgctcacg cctgtaatcc cagcactttg ggacaccgag4741 gtgggcagat cacttcaggt caggagtttg agaccagcct ggccaactta gtgaaacccc4801 atctctacta aaaatgcaaa aattatccag gcatggtggc acacgcctgt aatcccagct4861 ccacaggagg ctgaggcaga atcccttgaa gctgggaggc ggaggttgca gtgagccgag4921 attgcgccat tgcactccag cctgggcaac agagaaaaca aaaaggaaaa caaatgatga4981 aggtctgcag aaactgaaac ccagacatgt gtctgccccc tctatgtggg catggttttg5041 ccagtgcttc taagtgcagg agaacatgtc acctgaggct agttttgcat tcaggtccct5101 ggcttcgttt cttgttggta tgcctcccca gatcgtcctt cctgtatcca tgtgaccaga5161 ctgtatttgt tgggactgtc gcagatcttg gcttcttaca gttcttcctg tccaaactcc5221 atcctgtccc tcaggaacgg ggggaaaatt ctccgaatgt ttttggtttt ttggctgctt5281 ggaatttact tctgccacct gctggtcatc actgtcctca ctaagtggat tctggctccc5341 ccgtacctca tggctcaaac taccactcct cagtcgctat attaaagctt atattttgct5401 ggattactgc taaatacaaa agaaagttca atatgttttc atttctgtag ggaaaatggg5461 attgctgctt taaatttctg agctagggat tttttggcag ctgcagtgtt ggcgactatt5521 gtaaaattct ctttgtttct ctctgtaaat agcacctgct aacattacaa tttgtattta5581 tgtttaaaga aggcatcatt tggtgaacag aactaggaaa tgaattttta gctcttaaaa5641 gcatttgctt tgagaccgca caggagtgtc tttccttgta aaacagtgat gataatttct5701 gccttggccc taccttgaag caatgttgtg tgaagggatg aagaatctaa aagtcttcat5761 aagtccttgg gagaggtgct agaaaaatat aaggcactat cataattaca gtgatgtcct5821 tgctgttact actcaaatca cccacaaatt tccccaaaga ctgcgctagc tgtcaaataa5881 aagacagtga aattgacctg aaaaaaaaaa aaaaaaa

The Genbank ID for the TACC1 gene is 6867. Three isoforms are listed forTACC1, e.g., having Genebank Accession Nos. NP_(—)006274 (correspondingnucleotide sequence NM_(—)001174063); NP_(—)001167535 (correspondingnucleotide sequence NM_(—)001174064); NP_(—)001167536 (correspondingnucleotide sequence NM_(—)001174065).

SEQ ID NO: 148 is the TACC1 Amino Acid Sequence for isoform 1, havingGenebank Accession No. NP_(—)006274 (805 aa):

   1 MAFSPWQILS PVQWAKWTWS AVRGGAAGED EAGGPEGDPE EEDSQAETKS LSFSSDSEGN  61 FETPEAETPI RSPFKESCDP SLGLAGPGAK SQESQEADEQ LVAEVVEKCS SKTCSKPSEN 121 EVPQQAIDSH SVKNFREEPE HDFSKISIVR PFSIETKDST DISAVLGTKA AHGCVTAVSG 181 KALPSSPPDA LQDEAMTEGS MGVTLEASAE ADLKAGNSCP ELVPSRRSKL RKPKPVPLRK 241 KAIGGEFSDT NAAVEGTPLP KASYHFSPEE LDENTSPLLG DARFQKSPPD LKETPGTLSS 301 DTNDSGVELG EESRSSPLKL EFDFTEDTGN IEARKALPRK LGRKLGSTLT PKIQKDGISK 361 SAGLEQPTDP VARDGPLSQT SSKPDPSQWE SPSFNPFGSH SVLQNSPPLS SEGSYHFDPD 421 NFDESMDPFK PTTTLTSSDF CSPTGNHVNE ILESPKKAKS RLITSGCKVK KHETQSLALD 481 ACSRDEGAVI SQISDISNRD GHATDEEKLA STSCGQKSAG AEVKGEPEED LEYFECSNVP 541 VSTINHAFSS SEAGIEKETC QKMEEDGSTV LGLLESSAEK APVSVSCGGE SPLDGICLSE 601 SDKTAVLTLI REEIITKEIE ANEWKKKYEE TRQEVLEMRK IVAEYEKTIA QMIEDEQRTS 661 MTSQKSFQQL TMEKEQALAD LNSVERSLSD LFRRYENLKG VLEGFKKNEE ALKKCAQDYL 721 ARVKQEEQRY QALKIHAEEK LDKANEEIAQ VRTKAKAESA ALHAGLRKEQ MKVESLERAL 781 QQKNQEIEEL TKICDELIAK LGKTD

SEQ ID NO: 149 is the TACC1 Nucleotide Sequence for isoform 1, havingGenebank Accession No. NM_(—)006283 (7802 bp):

   1 agctgatgcg cgccccgccg gccgggaggc gggagtccgc gagccgggag cgggagcagc  61 agaggtctag cagccgggcg ccgcgggccg ggggcctgag gaggccacag gacgggcgtc 121 ttcccggcta gtggagcccg gcgcggggcc cgctgcggcc gcaccgtgag gggaggaggc 181 cgaggaggac gcagcgccgg ctgccggcgg gaggaagcgc tccaccaggg cccccgacgg 241 cactcgttta accacatccg cgcctctgct ggaaacgctt gctggcgcct gtcaccggtt 301 ccctccattt tgaaagggaa aaaggctctc cccacccatt cccctgcccc taggagctgg 361 agccggagga gccgcgctca tggcgttcag cccgtggcag atcctgtccc ccgtgcagtg 421 ggcgaaatgg acgtggtctg cggtacgcgg cggggccgcc ggcgaggacg aggctggcgg 481 gcccgagggc gaccccgagg aggaggattc gcaagccgag accaaatcct tgagtttcag 541 ctcggattct gaaggtaatt ttgagactcc tgaagctgaa accccgatcc gatcaccttt 601 caaggagtcc tgtgatccat cactcggatt ggcaggacct ggggccaaaa gccaagaatc 661 acaagaagct gatgaacagc ttgtagcaga agtggttgaa aaatgttcat ctaagacttg 721 ttctaaacct tcagaaaatg aagtgccaca gcaggccatt gactctcact cagtcaagaa 781 tttcagagaa gaacctgaac atgattttag caaaatttcc atcgtgaggc cattttcaat 841 agaaacgaag gattccacgg atatctcggc agtcctcgga acaaaagcag ctcatggctg 901 tgtaactgca gtctcaggca aggctctgcc ttccagcccg ccagacgccc tccaggacga 961 ggcgatgaca gaaggcagca tgggggtcac cctcgaggcc tccgcagaag ctgatctaaa1021 agctggcaac tcctgtccag agcttgtgcc cagcagaaga agcaagctga gaaagcccaa1081 gcctgtcccc ctgaggaaga aagcaattgg aggagagttc tcagacacca acgctgctgt1141 ggagggcaca cctctcccca aggcatccta tcacttcagt cctgaagagt tggatgagaa1201 cacaagtcct ttgctaggag atgccaggtt ccagaagtct ccccctgacc ttaaagaaac1261 tcccggcact ctcagtagtg acaccaacga ctcaggggtt gagctggggg aggagtcgag1321 gagctcacct ctcaagcttg agtttgattt cacagaagat acaggaaaca tagaggccag1381 gaaagccctt ccaaggaagc ttggcaggaa actgggtagc acactgactc ccaagataca1441 aaaagatggc atcagtaagt cagcaggttt agaacagcct acagacccag tggcacgaga1501 cgggcctctc tcccaaacat cttccaagcc agatcctagt cagtgggaaa gccccagctt1561 caaccccttt gggagccact ctgttctgca gaactcccca cccctctctt ctgagggctc1621 ctaccacttt gacccagata actttgacga atccatggat ccctttaaac caactacgac1681 cttaacaagc agtgactttt gttctcccac tggtaatcac gttaatgaaa tcttagaatc1741 acccaagaag gcaaagtcgc gtttaataac gagtggctgt aaggtgaaga agcatgaaac1801 tcagtctctc gccctggatg catgttctcg ggatgaaggg gcagtgatct cccagatttc1861 agacatttct aatagggatg gccatgctac tgatgaggag aaactggcat ccacgtcatg1921 tggtcagaaa tcagctggtg ccgaggtgaa aggtgagcca gaggaagacc tggagtactt1981 tgaatgttcc aatgttcctg tgtctaccat aaatcatgcg ttttcatcct cagaagcagg2041 catagagaag gagacgtgcc agaagatgga agaagacggg tccactgtgc ttgggctgct2101 ggagtcctct gcagagaagg cccctgtgtc ggtgtcctgt ggaggtgaga gccccctgga2161 tgggatctgc ctcagcgaat cagacaagac agccgtgctc accttaataa gagaagagat2221 aattactaaa gagattgaag caaatgaatg gaagaagaaa tacgaagaga cccggcaaga2281 agttttggag atgaggaaaa ttgtagctga atatgaaaag actattgctc aaatgattga2341 agatgaacaa aggacaagta tgacctctca gaagagcttc cagcaactga ccatggagaa2401 ggaacaggcc ctggctgacc ttaactctgt ggaaaggtcc ctttctgatc tcttcaggag2461 atatgagaac ctgaaaggtg ttctggaagg gttcaagaag aatgaagaag ccttgaagaa2521 atgtgctcag gattacttag ccagagttaa acaagaggag cagcgatacc aggccctgaa2581 aatccacgca gaagagaaac tggacaaagc caatgaagag attgctcagg ttcgaacaaa2641 agcaaaggct gagagtgcag ctctccatgc tggactccgc aaagagcaga tgaaggtgga2701 gtccctggaa agggccctgc agcagaagaa ccaagaaatt gaagaactga caaaaatctg2761 tgatgagctg attgcaaagc tgggaaagac tgac tgagac actccccctg ttagctcaac2821 agatctgcat ttggctgctt ctcttgtgac cacaattatc ttgccttatc caggaataat2881 tgcccctttg cagagaaaaa aaaaaactta aaaaaagcac atgcctactg ctgcctgtcc2941 cgctttgctg ccaatgcaac agccctggaa gaaaccctag agggttgcat agtctagaaa3001 ggagtgtgac ctgacagtgc tggagcctcc tagtttcccc ctatgaaggt tcccttaggc3061 tgctgagttt gggtttgtga tttatcttta gtttgtttta aagtcatctt tactttccca3121 aatgtgttaa atttgtaact cctctttggg gtcttctcca ccacctgtct gatttttttg3181 tgatctgttt aatcttttaa ttttttagta tcagtggttt tatttaagga gacagtttgg3241 cctattgtta cttccaattt ataatcaaga aggggctctg gatccccttt taaattacac3301 acactctcac acacatacat gtatgtttat agatgctgct gctcttttcc ctgaagcata3361 gtcaagtaag aactgctcta cagaaggaca tatttccttg gatgtgagac cctattttga3421 aatagagtcc tgactcagaa caccaactta agaatttggg ggattaaaga tgtgaagacc3481 acagtcttgg gttttcatat ctggagaaga ctatttgcca tgacgttttg ttgccctggt3541 atttggacac tcctcagctt taatgggtgt ggccccttta gggttagtcc tcagactaat3601 gatagtgtct gctttctgca tgaacggcaa tatgggactc cctccaagct agggtttggc3661 aagtctgccc tagagtcatt tactctcctc tgcctccatt tgttaataca gaatcaacat3721 ttagtcttca ttatcttttt tttttttttt gagacagagt ttcgatctat tttaagtatg3781 tgaagaaaat ctacttgtaa aaggctcaga tcttaattaa aaggtaattg tagcacatta3841 ccaattataa ggtgaagaaa tgtttttttc ccaagtgtga tgcattgttc ttcagatgtt3901 gaaaagaaag caaaaaatac cttctaactt aagacagaat ttttaacaaa atgagcagta3961 aaagtcacat gaaccactcc aaaaatcagt gcattttgca tatttttaaa caaagacagc4021 ttgttgaata ctgagaagag gagtgcaagg agaaggtctg tactaacaaa gccaaattcc4081 tcaagctctt actggactca gttcagagtg gtgggccatt aaccccaaca tggaattttt4141 ccatataaat ctcaatgaat tccctttcat ttgaataggc aaacccaaat ccatgcaagt4201 gttttaaagc actgtcctgt cttaatctta catgctgaaa gtcttcatgg tgatatgcac4261 tatattcagt atacgtatgt tttcctactt ctcttgtaaa actgttgcat gatccaactt4321 cagcaatgaa ttgtgcctag tggagaacct ctatagatct taaaaaatga attattcttt4381 agcagtgtat tactcacatg ggtgcaatct ttagccccag ggaggtcaat aatgtctttt4441 aaagccagaa gtcacatttt accaatatgc atttatcata attggtgctt aggctgtata4501 ttcaagcctg ttgtcttaac attttgtata aaaaagaaca acagaaatta tctgtcattt4561 gagaagtggc ttgacaatca tttgagcttt gaaagcagtc actgtggtgt aatatgaatg4621 ctgtcctagt ggtcatagta ccaagggcac gtgtctcccc ttggtataac tgatttcctt4681 tttagtcctc tactgctaaa taagttaatt ttgcattttg cagaaagaaa cattgattgc4741 taaatctttt tgctgctgtg ttttggtgtt ttcatgttta cttgttttat attgatctgt4801 tttaagtatg agaggcttat agtgccctcc attgtaaatc catagtcatc tttttaagct4861 tattgtgttt aagaaagtag ctatgtgtta aacagaggtg atggcagccc ttccctagca4921 cactggtgga agagacccct taagaacctg accccagtga atgaagctga tgcacaggga4981 gcaccaaagg accttcgtta agtgataatt gtcctggcct ctcagccatg accgttatga5041 ggaaatatcc cccattcgaa cttaacagat gcctcctctc caaagagaat taaaatcgta5101 gcttgtacag atcaagagaa tatactgggc agaatgaagt atgtttgttt atttttcttt5161 aaaaataaag gattttggaa ctctggagag taagaatata gtatagagtt tgcctcaaca5221 catgtgaggg ccaaataacc tgctagctag gcagtaataa actctgttac agaagagaaa5281 aagggccggg cacagtggct tattcctgta atcccaacac tgtggaaggc cgaggcagga5341 ggatcacttg agtccaggag tttgaaacct acctaggcaa catggtgaaa ccttgtctct5401 accaaaataa aaattagctg ggcatggtgg cacgtgcctg tggtcccagc tacttgggag5461 gctgaggtgg gagcctggga ggtcaaggct gcagtgagcc atgatcatgc cactgcactc5521 catcctgggt gacagcaaga tcttgtctca aaaaaaaaaa aaaaaaaaaa aaaaccagga5581 gtgaaaaagg aaagtagaag gcagctgctg gcctagatgt tggtttggga atattaggtg5641 atcctgttga gattctggat ccagagcaat ttctttagct tttgactttg ccaaagtgta5701 gatagccttt atccagcagt attttaagtg gggaatgcaa cgtgaggcca actgaacaat5761 tccccccgtg gctgcccaga tagtcacagt caaggttgga gagtctcctt ccagccagtg5821 acctacccaa accttttgtt ctgtaaaact gctctggaaa taccgggaag cccagttttc5881 tcacgtggtt tctagcttct tcagactcag cccaaattag gaagtgcaga agcacatgat5941 ggtgaaaaac ctaggatttg gcagccttcc agaatggtat ggaatctgag ggaagattta6001 tgtttcgttt tggaggatag ctcaagttga attttctttc cagccagtta ccctttcaac6061 ctacccatac tttgtacaac tcttacacaa atacttagat atttattaga tagccctgaa6121 ttcactctaa ttataaacag ggagtgtaaa ctgcccccag atgttcctgg gctgggtaaa6181 agcagctgga gtgaagcact cattttccat aaaggtaaca aagggcagct cagtggttac6241 tcaagctcaa aagggttttt ttaagagcaa gcattggtta agtctgtgta tactgagttg6301 gaagtgattt cagcacattc ttttttagtg gagtgaaagt tctgaagccc ccttttaact6361 tcctcttggt ttttcattat aattggtagc catctcatga actgtctctg actgttgtct6421 ctttgtggtc atgtgattgt gagcttgctt tctgacttgc atttctgact ttatcctgtt6481 gttaggaaga tagaaactag gttttgaaag attacatgat tcaagcgagg gattttaaag6541 taaagatgta tttattctga agaatctaaa agataacaga ttatttgctt atgaaagaac6601 aatatagtct gggaatccca gaatgtcaag ccaaaggtct aagaagtcat ctccttcaaa6661 tactttaata aagaagtatt tcgaggagat atctgtccaa aaaggtttga ctggcctcca6721 gattccagtt atttttaaaa agcaacttac cactaaatcc ttgagtctcc atagagtaac6781 agtaaagaaa ctgatgtaac agactctcct ctcaaaggat ctcctctgga agagactatc6841 agcggcagca ttctccaggg aagacccatc ccctagtgcc agagcttgca tcctggagac6901 taaagattgc acttttttgt agttttttgt ccaaatgcaa tcccatttct gtgcctctta6961 gcatgcagtt agatttggac aaacaagatt cctaaggaat gactttatta actataatat7021 ggttacagct attatataaa tatatattct ggttatagtt ctaatatgga gatgttgtgt7081 gcaatgctgg cctgtggtgg tctgtgtaat gctttaactt gtatggagga ggccaggctc7141 agagctgaga tgtggcctga accttccctg tatcgatcct ttaatttaga actgtcaaga7201 tgtcactttc tccccctctg ccttttagtg gtatctgaca tatactcaaa acagtaattt7261 cctggtcaca tcattaactg ctaattctgt atttataaag aattttcaga tggacatgta7321 caaatttgaa ctcaaaccat ccccagtcca gatacagggc agcgtgtagg tgaccacacc7381 agagcctcag cctcggtcct tctcagccgt cgggatagga tccaggcatt tcttttaaat7441 ctcagaggta gcagtaaact tttcagtatt gctgttagca agtgtgtgtt tgccaataga7501 tacccattat actaatgtgc caagtaaatg ttcattgcac atctgcttcc actgtgttcc7561 cacgggtgcc atgaagtgtg tgaggagccc ctcatctgga gggatgagtg ctgcgttgac7621 tactgctatc aggattgtgt tgtgtggaat attcatctac ataaatttta tatgcacagt7681 aatttccctt tttatatgtc aagtaactat ttgtaaaagt tatactcaca aattattata7741 atgattacta atatattttt tccatgtttc attgcctgaa taaaaactgt ttaccactgt7801 ta

SEQ ID NO: 150 is the amino acid sequence of the FGFR1-TACC1 fusionprotein.

MWSWKCLLFWAVLVTATLCTARPSPTLPEQDALPSSEDDDDDDDSSSEEKETDNTKPNPVAPYWTSPEKMEKKLHAVPAAKTVKFKCPSSGTPNPTLRWLKNGKEFKPDHRIGGYKVRYATWSIIMDSVVPSDKGNYTCIVENEYGSINHTYQLDVVERSPHRPILQAGLPANKTVALGSNVEFMCKVYSDPQPHIQWLKHIEVNGSKIGPDNLPYVQILKTAGVNTTDKEMEVLHLRNVSFEDAGEYTCLAGNSIGLSHHSAWLTVLEALEERPAVMTSPLYLEIIIYCTGAFLISCMVGSVIVYKMKSGTKKSDFHSQMAVHKLAKSIPLRRQVTVSADSSASMNSGVLLVRPSRLSSSGTPMLAGVSEYELPEDPRWELPRDRLVLGKPLGEGCFGQVVLAEAIGLDKDKPNRVTKVAVKMLKSDATEKDLSDLISEMEMMKMIGKHKNIINLLGACTQDGPLYVIVEYASKGNLREYLQARRPPGLEYCYNPSHNPEEQLSSKDLVSCAYQVARGMEYLASKKCIHRDLAARNVLVTEDNVMKIADFGLARDIHHIDYYKKTTNGRLPVKWMAPEALFDRIYTHQSDVWSFGVLLWEIFTLGGSPYPGVPVEELFKLLKEGHRMDKPSNCTNELYMMMRDCWHAVPSQRPTFKQLVEDLDRIVALTSNQGLLESSAEKAPVSVSCGGESPLDGICLSESDKTAVLTLIREEIITKEIEANEWKKKYEETRQEVLEMRKIVAEYEKTIAQMIEDEQRTSMTSQKSFQQLTMEKEQALADLNSVERSLSDLFRRYENLKGVLEGFKKNEEALKKCAQDYLARVKQEEQRYQALKIHAEEKLDKANEEIAQVRTKAKAESAALHAGLRKEQMKVESLERALQQKNQEIEELTKICDELI AKLGKTD

SEQ ID NO: 151 is the nucleotide sequence that encodes the FGFR1-TACC1fusion protein.

atgtggagctggaagtgcctcctcttctgggctgtgctggtcacagccacactctgcaccgctaggccgtccccgaccttgcctgaacaagcccagccctggggagcccctgtggaagtggagtccttcctggtccaccccggtgacctgctgcagcttcgctgtcggctgcgggacgatgtgcagagcatcaactggctgcgggacggggtgcagctggcggaaagcaaccgcacccgcatcacaggggaggaggtggaggtgcaggactccgtgcccgcagactccggcctctatgcttgcgtaaccagcagcccctcgggcagtgacaccacctacttctccgtcaatgtttcagatgctctcccctcctcggaggatgatgatgatgatgatgactcctcttcagaggagaaagaaacagataacaccaaaccaaaccgtatgcccgtagctccatattggacatccccagaaaagatggaaaagaaattgcatgcagtgccggctgccaagacagtgaagttcaaatgcccttccagtgggaccccaaaccccacactgcgctggttgaaaaatggcaaagaattcaaacctgaccacagaattggaggctacaaggtccgttatgccacctggagcatcataatggactctgtggtgccctctgacaagggcaactacacctgcattgtggagaatgagtacggcagcatcaaccacacataccagctggatgtcgtggagcggtcccctcaccggcccatcctgcaagcagggttgcccgccaacaaaacagtggccctgggtagcaacgtggagttcatgtgtaaggtgtacagtgacccgcagccgcacatccagtggctaaagcacatcgaggtgaatgggagcaagattggcccagacaacctgccttatgtccagatcttgaagactgctggagttaataccaccgacaaagagatggaggtgcttcacttaagaaatgtctcctttgaggacgcaggggagtatacgtgcttggcgggtaactctatcggactctcccatcactctgcatggttgaccgttctggaagccctggaagagaggccggcagtgatgacctcgcccctgtacctggagatcatcatctattgcacaggggccttcctcatctcctgcatggtggggtcggtcatcgtctacaagatgaagagtggtaccaagaagagtgacttccacagccagatggctgtgcacaagctggccaagagcatccctctgcgcagacaggtgtctgctgactccagtgcatccatgaactctggggttcttctggttcggccatcacggctctcctccagtgggactcccatgctagcaggggtctctgagtatgagcttcccgaagaccctcgctgggagctgcctcgggacagactggtcttaggcaaacccctgggagagggctgctttgggcaggtggtgttggcagaggctatcgggctggacaaggacaaacccaaccgtgtgaccaaagtggctgtgaagatgttgaagtcggacgcaacagagaaagacttgtcagacctgatctcagaaatggagatgatgaagatgatcgggaagcataagaatatcatcaacctgctgggggcctgcacgcaggatggtcccttgtatgtcatcgtggagtatgcctccaagggcaacctgcgggagtacctgcaggcccggaggcccccagggctggaatactgctacaaccccagccacaacccagaggagcagctctcctccaaggacctggtgtcctgcgcctaccaggtggcccgaggcatggagtatctggcctccaagaagtgcatacaccgagacctggcagccaggaatgtcctggtgacagaggacaatgtgatgaagatagcagactttggcctcgcacgggacattcaccacatcgactactataaaaagacaaccaacggccgactgcctgtgaagtggatggcacccgaggcattatttgaccggatctacacccaccagagtgatgtgtggtctttcggggtgctcctgtgggagatcttcactctgggcggctccccataccccggtgtgcctgtggaggaacttttcaagctgctgaaggagggtcaccgcatggacaagcccagtaactgcaccaacgagctgtacatgatgatgcgggactgctggcatgcagtgccctcacagagacccaccttcaagcagctggtggaagacctggaccgcatcgtggccttgacctccaaccagtgggctgctggagtcctctgcagagaaggcccctgtgtcggtgtcctgtggaggtgagagccccctggatgggatctgcctcagcgaatcagacaagacagccgtgctcaccttaataagagaagagataattactaaagagattgaagcaaatgaatggaagaagaaatacgaagagacccggcaagaagttttggagatgaggaaaattgtagctgaatatgaaaagactattgctcaaatgattgaagatgaacaaaggacaagtatgacctctcagaagagcttccagcaactgaccatggagaaggaacaggccctggctgaccttaactctgtggaaaggtccctttctgatctcttcaggagatatgagaacctgaaaggtgttctggaagggttcaagaagaatgaagaagccttgaagaaatgtgctcaggattacttagccagagttaaacaagaggagcagcgataccaggccctgaaaatccacgcagaagagaaactggacaaagccaatgaagagattgctcaggttcgaacaaaagcaaaggctgagagtgcagctctccatgctggactccgcaaagagcagatgaaggtggagtccctggaaagggccctgcagcagaagaaccaagaaattgaagaactgacaaaaatctgtgatgagctgattgc aaagctgggaaagactgac

The Genbank ID for the FGFR2 gene is 2263. Eight isoforms are listed forFGFR2, e.g., having Genebank Accession Nos. NP_(—)000132 (correspondingnucleotide sequence NM_(—)000141); NP_(—)001138385 (correspondingnucleotide sequence NM_(—)001144913); NP_(—)001138386 (correspondingnucleotide sequence NM_(—)001144914); NP_(—)001138387 (correspondingnucleotide sequence NM_(—)001144915); NP_(—)001138388 (correspondingnucleotide sequence NM_(—)001144916); NP_(—)001138389 (correspondingnucleotide sequence NM_(—)001144917); NP_(—)001138390 (correspondingnucleotide sequence NM_(—)001144918); NP_(—)001138391 (correspondingnucleotide sequence NM_(—)001144919); NP_(—)075259 (correspondingnucleotide sequence NM_(—)022970).

SEQ ID NO: 152 is the FGFR2 Amino Acid Sequence for isoform 1, havingGenebank Accession No. NP_(—)000132 (821 aa):

  1 MVSWGRFICL VVVTMATLSL ARPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV 61 RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF121 MVNVTDAISS GDDEDDTDGA EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP181 AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI241 NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK301 YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL361 PAPGREKEIT ASPDYLEIAI YCIGVFLIAC MVVTVILCRM KNTTKKPDFS SQPAVHKLTK421 RIPLRRQVTV SAESSSSMNS NTPLVRITTR LSSTADTPML AGVSEYELPE DPKWEFPRDK481 LTLGKPLGEG CFGQVVMAEA VGIDKDKPKE AVTVAVKMLK DDATEKDLSD LVSEMEMMKM541 IGKHKNIINL LGACTQDGPL YVIVEYASKG NLREYLRARR PPGMEYSYDI NRVPEEQMTF601 KDLVSCTYQL ARGMEYLASQ KCIHRDLAAR NVLVTENNVM KIADFGLARD INNIDYYKKT661 TNGRLPVKWM APEALFDRVY THQSDVWSFG VLMWEIFTLG GSPYPGIPVE ELFKLLKEGH721 RMDKPANCTN ELYMMMRDCW HAVPSQRPTF KQLVEDLDRI LTLTTNEEYL DLSQPLEQYS781 PSYPDTRSSC SSGDDSVFSP DPMPYEPCLP QYPHINGSVK T

SEQ ID NO: 153 is the FGFR2 Nucleotide Sequence for isoform 1, havingGenebank Accession No. NM_(—)000141 (4654 bp):

   1 ggcggcggct ggaggagagc gcggtggaga gccgagcggg cgggcggcgg gtgcggagcg  61 ggcgagggag cgcgcgcggc cgccacaaag ctcgggcgcc gcggggctgc atgcggcgta 121 cctggcccgg cgcggcgact gctctccggg ctggcggggg ccggccgcga gccccggggg 181 ccccgaggcc gcagcttgcc tgcgcgctct gagccttcgc aactcgcgag caaagtttgg 241 tggaggcaac gccaagcctg agtcctttct tcctctcgtt ccccaaatcc gagggcagcc 301 cgcgggcgtc atgcccgcgc tcctccgcag cctggggtac gcgtgaagcc cgggaggctt 361 ggcgccggcg aagacccaag gaccactctt ctgcgtttgg agttgctccc cgcaaccccg 421 ggctcgtcgc tttctccatc ccgacccacg cggggcgcgg ggacaacaca ggtcgcggag 481 gagcgttgcc attcaagtga ctgcagcagc agcggcagcg cctcggttcc tgagcccacc 541 gcaggctgaa ggcattgcgc gtagtccatg cccgtagagg aagtgtgcag atgggattaa 601 cgtccacatg gagatatgga agaggaccgg ggattggtac cgtaaccatg gtcagctggg 661 gtcgtttcat ctgcctggtc gtggtcacca tggcaacctt gtccctggcc cggccctcct 721 tcagtttagt tgaggatacc acattagagc cagaagagcc accaaccaaa taccaaatct 781 ctcaaccaga agtgtacgtg gctgcgccag gggagtcgct agaggtgcgc tgcctgttga 841 aagatgccgc cgtgatcagt tggactaagg atggggtgca cttggggccc aacaatagga 901 cagtgcttat tggggagtac ttgcagataa agggcgccac gcctagagac tccggcctct 961 atgcttgtac tgccagtagg actgtagaca gtgaaacttg gtacttcatg gtgaatgtca1021 cagatgccat ctcatccgga gatgatgagg atgacaccga tggtgcggaa gattttgtca1081 gtgagaacag taacaacaag agagcaccat actggaccaa cacagaaaag atggaaaagc1141 ggctccatgc tgtgcctgcg gccaacactg tcaagtttcg ctgcccagcc ggggggaacc1201 caatgccaac catgcggtgg ctgaaaaacg ggaaggagtt taagcaggag catcgcattg1261 gaggctacaa ggtacgaaac cagcactgga gcctcattat ggaaagtgtg gtcccatctg1321 acaagggaaa ttatacctgt gtagtggaga atgaatacgg gtccatcaat cacacgtacc1381 acctggatgt tgtggagcga tcgcctcacc ggcccatcct ccaagccgga ctgccggcaa1441 atgcctccac agtggtcgga ggagacgtag agtttgtctg caaggtttac agtgatgccc1501 agccccacat ccagtggatc aagcacgtgg aaaagaacgg cagtaaatac gggcccgacg1561 ggctgcccta cctcaaggtt ctcaaggccg ccggtgttaa caccacggac aaagagattg1621 aggttctcta tattcggaat gtaacttttg aggacgctgg ggaatatacg tgcttggcgg1681 gtaattctat tgggatatcc tttcactctg catggttgac agttctgcca gcgcctggaa1741 gagaaaagga gattacagct tccccagact acctggagat agccatttac tgcatagggg1801 tcttcttaat cgcctgtatg gtggtaacag tcatcctgtg ccgaatgaag aacacgacca1861 agaagccaga cttcagcagc cagccggctg tgcacaagct gaccaaacgt atccccctgc1921 ggagacaggt aacagtttcg gctgagtcca gctcctccat gaactccaac accccgctgg1981 tgaggataac aacacgcctc tcttcaacgg cagacacccc catgctggca ggggtctccg2041 agtatgaact tccagaggac ccaaaatggg agtttccaag agataagctg acactgggca2101 agcccctggg agaaggttgc tttgggcaag tggtcatggc ggaagcagtg ggaattgaca2161 aagacaagcc caaggaggcg gtcaccgtgg ccgtgaagat gttgaaagat gatgccacag2221 agaaagacct ttctgatctg gtgtcagaga tggagatgat gaagatgatt gggaaacaca2281 agaatatcat aaatcttctt ggagcctgca cacaggatgg gcctctctat gtcatagttg2341 agtatgcctc taaaggcaac ctccgagaat acctccgagc ccggaggcca cccgggatgg2401 agtactccta tgacattaac cgtgttcctg aggagcagat gaccttcaag gacttggtgt2461 catgcaccta ccagctggcc agaggcatgg agtacttggc ttcccaaaaa tgtattcatc2521 gagatttagc agccagaaat gttttggtaa cagaaaacaa tgtgatgaaa atagcagact2581 ttggactcgc cagagatatc aacaatatag actattacaa aaagaccacc aatgggcggc2641 ttccagtcaa gtggatggct ccagaagccc tgtttgatag agtatacact catcagagtg2701 atgtctggtc cttcggggtg ttaatgtggg agatcttcac tttagggggc tcgccctacc2761 cagggattcc cgtggaggaa ctttttaagc tgctgaagga aggacacaga atggataagc2821 cagccaactg caccaacgaa ctgtacatga tgatgaggga ctgttggcat gcagtgccct2881 cccagagacc aacgttcaag cagttggtag aagacttgga tcgaattctc actctcacaa2941 ccaatgagga atacttggac ctcagccaac ctctcgaaca gtattcacct agttaccctg3001 acacaagaag ttcttgttct tcaggagatg attctgtttt ttctccagac cccatgcctt3061 acgaaccatg ccttcctcag tatccacaca taaacggcag tgttaaaaca tgaatgactg3121 tgtctgcctg tccccaaaca ggacagcact gggaacctag ctacactgag cagggagacc3181 atgcctccca gagcttgttg tctccacttg tatatatgga tcagaggagt aaataattgg3241 aaaagtaatc agcatatgtg taaagattta tacagttgaa aacttgtaat cttccccagg3301 aggagaagaa ggtttctgga gcagtggact gccacaagcc accatgtaac ccctctcacc3361 tgccgtgcgt actggctgtg gaccagtagg actcaaggtg gacgtgcgtt ctgccttcct3421 tgttaatttt gtaataattg gagaagattt atgtcagcac acacttacag agcacaaatg3481 cagtatatag gtgctggatg tatgtaaata tattcaaatt atgtataaat atatattata3541 tatttacaag gagttatttt ttgtattgat tttaaatgga tgtcccaatg cacctagaaa3601 attggtctct ctttttttaa tagctatttg ctaaatgctg ttcttacaca taatttctta3661 attttcaccg agcagaggtg gaaaaatact tttgctttca gggaaaatgg tataacgtta3721 atttattaat aaattggtaa tatacaaaac aattaatcat ttatagtttt ttttgtaatt3781 taagtggcat ttctatgcag gcagcacagc agactagtta atctattgct tggacttaac3841 tagttatcag atcctttgaa aagagaatat ttacaatata tgactaattt ggggaaaatg3901 aagttttgat ttatttgtgt ttaaatgctg ctgtcagacg attgttctta gacctcctaa3961 atgccccata ttaaaagaac tcattcatag gaaggtgttt cattttggtg tgcaaccctg4021 tcattacgtc aacgcaacgt ctaactggac ttcccaagat aaatggtacc agcgtcctct4081 taaaagatgc cttaatccat tccttgagga cagaccttag ttgaaatgat agcagaatgt4141 gcttctctct ggcagctggc cttctgcttc tgagttgcac attaatcaga ttagcctgta4201 ttctcttcag tgaattttga taatggcttc cagactcttt ggcgttggag acgcctgtta4261 ggatcttcaa gtcccatcat agaaaattga aacacagagt tgttctgctg atagttttgg4321 ggatacgtcc atctttttaa gggattgctt tcatctaatt ctggcaggac ctcaccaaaa4381 gatccagcct catacctaca tcagacaaaa tatcgccgtt gttccttctg tactaaagta4441 ttgtgttttg ctttggaaac acccactcac tttgcaatag ccgtgcaaga tgaatgcaga4501 ttacactgat cttatgtgtt acaaaattgg agaaagtatt taataaaacc tgttaatttt4561 tatactgaca ataaaaatgt ttctacagat attaatgtta acaagacaaa ataaatgtca4621 cgcaacttat ttttttaata aaaaaaaaaa aaaa

The Genbank ID for the TACC2 gene is 10579. Four isoforms are listed forTACC2, e.g., having Genebank Accession Nos. NP_(—)996744 (correspondingnucleotide sequence NM_(—)206862); NP_(—)996743 (correspondingnucleotide sequence NM_(—)206861); NP_(—)996742 (correspondingnucleotide sequence NM_(—)206860); NP_(—)008928 (correspondingnucleotide sequence NM_(—)006997).

SEQ ID NO: 154 is the TACC2 Amino Acid Sequence for isoform a, havingGenebank Accession No. NP_(—)996744 (2948 aa):

   1 MGNENSTSDN QRTLSAQTPR SAQPPGNSQN IKRKQQDTPG SPDHRDASSI GSVGLGGFCT  61 ASESSASLDP CLVSPEVTEP RKDPQGARGP EGSLLPSPPP SQEREHPSSS MPFAECPPEG 121 CLASPAAAPE DGPQTQSPRR EPAPNAPGDI AAAFPAERDS STPYQEIAAV PSAGRERQPK 181 EEGQKSSFSF SSGIDQSPGM SPVPLREPMK APLCGEGDQP GGFESQEKEA AGGFPPAESR 241 QGVASVQVTP EAPAAAQQGT ESSAVLEKSP LKPMAPIPQD PAPRASDRER GQGEAPPQYL 301 TDDLEFLRAC HLPRSNSGAA PEAEVNAASQ ESCQQPVGAY LPHAELPWGL PSPALVPEAG 361 GSGKEALDTI DVQGHPQTGM RGTKPNQVVC VAAGGQPEGG LPVSPEPSLL TPTEEAHPAS 421 SLASFPAAQI PIAVEEPGSS SRESVSKAGM PVSADAAKEV VDAGLVGLER QVSDLGSKGE 481 HPEGDPGEVP APSPQERGEH LNTEQSHEVQ PGVPPPPLPK EQSHEVQPGA PPPPLPKAPS 541 ESARGPPGPT DGAKVHEDST SPAVAKEGSR SPGDSPGGKE EAPEPPDGGD PGNLQGEDSQ 601 AFSSKRDPEV GKDELSKPSS DAESRDHPSS HSAQPPRKGG AGHTDGPHSQ TAEADASGLP 661 HKLGEEDPVL PPVPDGAGEP TVPEGAIWEG SGLQPKCPDT LQSREGLGRM ESFLTLESEK 721 SDFPPTPVAE VAPKAQEGES TLEIRKMGSC DGEGLLTSPD QPRGPACDAS RQEFHAGVPH 781 PPQGENLAAD LGLTALILDQ DQQGIPSCPG EGWIRGAASE WPLLSSEKHL QPSQAQPETS 841 IFDVLKEQAQ PPENGKETSP SHPGFKDQGA DSSQIHVPVE PQEDNNLPTH GGQEQALGSE 901 LQSQLPKGTL SDTPTSSPTD MVWESSLTEE SELSAPTRQK LPALGEKRPE GACGDGQSSR 961 VSPPAADVLK DFSLAGNFSR KETCCTGQGP NKSQQALADA LEEGSQHEEA CQRHPGASEA1021 ADGCSPLWGL SKREMASGNT GEAPPCQPDS VALLDAVPCL PALAPASPGV TPTQDAPETE1081 ACDETQEGRQ QPVPAPQQKM ECWATSDAES PKLLASFPSA GEQGGEAGAA ETGGSAGAGD1141 PGKQQAPEKP GEATLSCGLL QTEHCLTSGE EASTSALRES CQAEHPMASC QDALLPAREL1201 GGIPRSTMDF STHQAVPDPK ELLLSGPPEV AAPDTPYLHV DSAAQRGAED SGVKAVSSAD1261 PRAPGESPCP VGEPPLALEN AASLKLFAGS LAPLLQPGAA GGEIPAVQAS SGSPKARTTE1321 GPVDSMPCLD RMPLLAKGKQ ATGEEKAATA PGAGAKASGE GMAGDAAGET EGSMERMGEP1381 SQDPKQGTSG GVDTSSEQIA TLTGFPDFRE HIAKIFEKPV LGALATPGEK AGAGRSAVGK1441 DLTRPLGPEK LLDGPPGVDV TLLPAPPARL QVEKKQQLAG EAEISHLALQ DPASDKLLGP1501 AGLTWERNLP GAGVGKEMAG VPPTLREDER PEGPGAAWPG LEGQAYSQLE RSRQELASGL1561 PSPAATQELP VERAAAFQVA PHSHGEEAVA QDRIPSGKQH QETSACDSPH GEDGPGDFAH1621 TGVPGHVPRS TCAPSPQREV LTVPEANSEP WTLDTLGGER RPGVTAGILE MRNALGNQST1681 PAPPTGEVAD TPLEPGKVAG AAGEAEGDIT LSTAETQACA SGDLPEAGTT RTFSVVAGDL1741 VLPGSCQDPA CSDKAPGMEG TAALHGDSPA RPQQAKEQPG PERPIPAGDG KVCVSSPPEP1801 DETHDPKLQH LAPEELHTDR ESPRPGPSML PSVPKKDAPR VMDKVTSDET RGAEGTESSP1861 VADDIIQPAA PADLESPTLA ASSYHGDVVG QVSTDLIAQS ISPAAAHAGL PPSAAEHIVS1921 PSAPAGDRVE ASTPSCPDPA KDLSRSSDSE EAFETPESTT PVKAPPAPPP PPPEVIPEPE1981 VSTQPPPEEP GCGSETVPVP DGPRSDSVEG SPFRPPSHSF SAVFDEDKPI ASSGTYNLDF2041 DNIELVDTFQ TLEPRASDAK NQEGKVNTRR KSTDSVPISK STLSRSLSLQ ASDFDGASSS2101 GNPEAVALAP DAYSTGSSSA SSTLKRTKKP RPPSLKKKQT TKKPTETPPV KETQQEPDEE2161 SLVPSGENLA SETKTESAKT EGPSPALLEE TPLEPAVGPK AACPLDSESA EGVVPPASGG2221 GRVQNSPPVG RKTLPLTTAP EAGEVTPSDS GGQEDSPAKG LSVRLEFDYS EDKSSWDNQQ2281 ENPPPTKKIG KKPVAKMPLR RPKMKKTPEK LDNTPASPPR SPAEPNDIPI AKGTYTFDID2341 KWDDPNFNPF SSTSKMQESP KLPQQSYNFD PDTCDESVDP FKTSSKTPSS PSKSPASFEI2401 PASAMEANGV DGDGLNKPAK KKKTPLKTDT FRVKKSPKRS PLSDPPSQDP TPAATPETPP2461 VISAVVHATD EEKLAVTNQK WTCMTVDLEA DKQDYPQPSD LSTFVNETKF SSPTEELDYR2521 NSYEIEYMEK IGSSLPQDDD APKKQALYLM FDTSQESPVK SSPVRMSESP TPCSGSSFEE2581 TEALVNTAAK NQHPVPRGLA PNQESHLQVP EKSSQKELEA MGLGTPSEAI EITAPEGSFA2641 SADALLSRLA HPVSLCGALD YLEPDLAEKN PPLFAQKLQE ELEFAIMRIE ALKLARQIAL2701 ASRSHQDAKR EAAHPTDVSI SKTALYSRIG TAEVEKPAGL LFQQPDLDSA LQIARAEIIT2761 KEREVSEWKD KYEESRREVM EMRKIVAEYE KTIAQMIEDE QREKSVSHQT VQQLVLEKEQ2821 ALADLNSVEK SLADLFRRYE KMKEVLEGFR KNEEVLKRCA QEYLSRVKKE EQRYQALKVH2881 AEEKLDRANA EIAQVRGKAQ QEQAAHQASL RKEQLRVDAL ERTLEQKNKE IEELTKICDE2941 LIAKMGKS

SEQ ID NO: 155 is the TACC2 Nucleotide Sequence for isoform a, havingGenebank Accession No. NM_(—)206862 (9706 bp)

   1 gcctgctcca agggaaggat caggagagaa gaaacgcaaa tcccagaacc gtgccaacat  61 ataaaacccc acattaaggg ttgtacagtg cactgggatt tctcaagtca cccgcttggt 121 cctcttccaa gtatacttta cttcctttca ttcctctcta aaactttttt aaaaactttc 181 actcctgctc taaaagttat cttggtttct tactctacct tatgcccctt gggcgaattt 241 tttcctctga ggagggaaga atagagttgc tgctgcagac acatcagatt ccctactggt 301 aacagctgga gtgcgtcacc tctgacaaaa ttctggggac gctgggaaca ctgaatcaac 361 atgggcaatg agaacagcac ctcggacaac cagaggactt tatcagctca gactccaagg 421 tccgcgcagc cacccgggaa cagtcagaat ataaaaagga agcagcagga cacgcccgga 481 agccctgacc acagagacgc gtccagcatt ggcagcgttg ggcttggagg cttctgcacc 541 gcttctgaga gttctgccag cctggatcca tgccttgtgt ccccagaggt gactgagcca 601 aggaaggacc cacagggagc cagggggcca gaaggttctt tgctgcccag cccaccaccg 661 tcccaggagc gagagcaccc ctcgtcctcc atgccctttg ccgagtgtcc cccggaaggt 721 tgcttggcaa gtccagcagc ggcacctgaa gatggtcctc agactcagtc tcccaggagg 781 gaacctgccc caaatgcccc aggagacatc gcggcggcat ttcccgctga gagggacagc 841 tctactccat accaagagat tgctgccgtc cccagtgctg gaagagagag acagccgaag 901 gaagaaggac agaagtcctc cttctccttc tccagtggca tcgaccagtc acctggaatg 961 tcgccagtac ccctcagaga gccaatgaag gcaccgctgt gtggagaggg ggaccagcct1021 ggtggttttg agtcccaaga gaaagaggct gcaggtggct ttccccctgc agagtccagg1081 cagggggtgg cttctgtgca agtgacccct gaggcccctg ctgcagccca gcagggcaca1141 gaaagctcag cggtcttgga gaagtccccc ctaaaaccca tggccccgat cccacaagat1201 ccagccccaa gagcctcaga cagagaaaga ggccaagggg aggcgccgcc tcagtattta1261 acagatgact tggaattcct cagggcctgc catctcccta ggagcaattc aggggctgcc1321 ccagaagcag aagtgaatgc cgcttcccag gagagctgcc agcagccagt gggagcatat1381 ctgccgcacg cagagctgcc ctggggcttg ccaagtcctg ccctggtgcc agaggctggg1441 ggctctggga aggaggctct ggacaccatt gatgttcagg gtcacccaca gacagggatg1501 cgaggaacca agcccaatca agttgtctgt gtggcagcag gcggccagcc cgaagggggt1561 ttgcctgtga gccctgaacc ttccctgctc actccgactg aggaagcaca tccagcttca1621 agcctcgctt cattcccagc tgctcagatt cctattgctg tagaagaacc tggatcatca1681 tccagggaat cagtttccaa ggctgggatg ccagtttctg cagatgcagc caaagaggtg1741 gtggatgcag ggttggtggg actggagagg caggtgtcag atcttggaag caagggagag1801 catccagaag gggaccctgg agaggttcct gccccatcac cccaggagag gggagagcac1861 ttgaacacgg agcaaagcca tgaggtccaa ccaggagtac caccccctcc tcttcccaag1921 gagcaaagcc atgaggtcca accaggagca ccaccccctc ctcttcccaa ggcaccaagt1981 gaaagtgcca gagggccacc ggggccaacg gatggagcca aggtccatga agattccaca2041 agcccagccg tggctaaaga aggaagcaga tcacctggtg acagccctgg aggaaaggag2101 gaagccccag agccacctga tggtggagac ccagggaacc tgcaaggaga ggactctcag2161 gctttcagca gcaagcgtga tccagaagta ggcaaagatg agctttcaaa gccaagcagt2221 gatgcagaga gcagagacca tcccagctca cactcagcac agccacccag aaaggggggt2281 gctgggcaca cggacgggcc ccactctcag acagcagagg ctgatgcatc tggcctacca2341 cacaagctgg gtgaggagga ccccgtcctg ccccctgtgc cagatggagc tggtgagccc2401 actgttcccg aaggagccat ctgggagggg tcaggattgc agcccaaatg tcctgacacc2461 cttcagagca gggaaggatt gggaagaatg gagtctttcc tgactttaga atcagagaaa2521 tcagattttc caccaactcc tgttgcagag gttgcaccca aagcccagga aggtgagagc2581 acattggaaa taaggaagat gggcagctgt gatggggagg gcttgctgac gtccccagat2641 caaccccgcg ggccggcgtg tgatgcgtcg agacaggaat ttcatgctgg ggtgccacat2701 cccccccagg gggagaactt ggcagcagac ctggggctca cggcactcat cctggaccaa2761 gatcagcagg gaatcccatc ctgcccaggg gaaggctgga taagaggagc tgcatccgag2821 tggcccctac tatcttctga gaagcatctc cagccatccc aggcacaacc agagacatcc2881 atctttgacg tgctcaagga gcaggcccag ccacctgaaa atgggaaaga gacttctcca2941 agccatccag gttttaagga ccagggagca gattcttccc aaatccatgt acctgtggaa3001 cctcaggaag ataacaactt gcccactcat ggaggacagg agcaggcttt gggatcagaa3061 cttcaaagtc agctccccaa aggcaccctg tctgatactc caacttcatc tcccactgac3121 atggtttggg agagttctct gacagaagag tcagaattgt cagcaccaac gagacagaag3181 ttgcctgcac taggggagaa gcggccagag ggagcatgcg gtgatggtca gtcctcgagg3241 gtctcgcctc cagcagcaga tgtcttaaaa gacttttctc ttgcagggaa cttcagcaga3301 aaggaaactt gctgcactgg gcaggggcca aacaagtctc aacaggcatt ggctgatgcc3361 ttggaagaag gcagccagca tgaagaagca tgtcaaaggc atccaggagc ttctgaagca3421 gctgatggtt gttccccact ctggggcttg agtaagaggg agatggcaag tggaaacaca3481 ggggaggccc caccttgtca gcctgactca gtagctctcc tggatgcagt tccctgcctg3541 ccagccctgg cgcccgccag ccccggagtc acacccaccc aggatgcccc agagacagag3601 gcatgtgatg aaacccagga aggcaggcag caaccagtgc cggccccgca gcagaaaatg3661 gagtgctggg ccacttcgga tgcagagtcc ccaaagcttc ttgcaagttt cccatcagct3721 ggggagcaag gtggtgaagc cggggctgct gagactggtg gcagcgctgg tgcaggagac3781 ccaggaaagc agcaggctcc ggagaaacct ggagaagcta ctttgagttg tggcctcctt3841 cagactgagc actgccttac ctccggggag gaagcttcta cctctgccct acgtgagtcc3901 tgccaagctg agcaccccat ggccagctgc caggatgcct tgctgccagc cagagagctg3961 ggtgggattc ccaggagcac catggatttt tctacacacc aggctgtccc agacccaaag4021 gagctcctgc tgtctgggcc accagaagtg gctgctcctg acacccctta cctgcatgtc4081 gacagtgctg cccagagagg agcagaagac agtggagtga aagctgtttc ctctgcagac4141 cccagagctc ctggcgaaag cccctgtcct gtaggggagc ccccacttgc cttggaaaat4201 gctgcctcct tgaagctgtt tgctggctcc ctcgcccccc tgttgcaacc aggagctgca4261 ggtggggaaa tccctgcagt gcaagccagc agtggtagtc ccaaagccag aaccactgag4321 ggaccagtgg actccatgcc atgcctggac cggatgccac ttctggccaa gggcaagcag4381 gcaacagggg aagagaaagc agcaacagct ccaggtgcag gtgccaaggc cagtggggag4441 ggcatggcag gtgatgcagc aggagagaca gagggcagca tggagaggat gggagagcct4501 tcccaggacc caaagcaggg cacatcaggt ggtgtggaca caagctctga gcaaatcgcc4561 accctcactg gcttcccaga cttcagggag cacatcgcca agatcttcga gaagcctgtg4621 ctcggagccc tggccacacc tggagaaaag gcaggagctg ggaggagtgc agtgggtaaa4681 gacctcacca ggccattggg cccagagaag cttctagatg ggcctccagg agtggatgtc4741 acccttctcc ctgcacctcc tgctcgactc caggtggaga agaagcaaca gttggctgga4801 gaggctgaga tttcccatct ggctctgcaa gatccagctt cagacaagct tctgggtcca4861 gcagggctga cctgggagcg gaacttgcca ggtgccggtg tggggaagga gatggcaggt4921 gtcccaccca cactgaggga agacgagagg ccagaggggc ctggggcagc ctggccaggc4981 ctggaaggcc aggcttactc acagctggag aggagcaggc aggaattagc ttcaggtctt5041 ccttcaccag cagctactca ggagctccct gtggagagag ctgctgcctt ccaggtggct5101 ccccatagcc atggagaaga ggccgtggcc caagacagaa ttccttctgg aaagcagcac5161 caggaaacat ctgcctgcga cagtccacat ggagaagatg gtcccgggga ctttgctcac5221 acaggggttc caggacatgt gccaaggtcc acgtgtgccc cttctcctca gagggaggtt5281 ttgactgtgc ctgaggccaa cagtgagccc tggacccttg acacgcttgg gggtgaaagg5341 agacccggag tcactgctgg catcttggaa atgcgaaatg ccctgggcaa ccagagcacc5401 cctgcaccac caactggaga agtggcagac actcccctgg agcctggcaa ggtggcaggc5461 gctgctgggg aagcagaggg tgacatcacc ctgagcacag ctgagacaca ggcatgtgcg5521 tccggtgatc tgcctgaagc aggtactacg aggacattct ccgttgtggc aggtgacttg5581 gtgctgccag gaagctgtca ggacccagcc tgctctgaca aggctccggg gatggagggt5641 acagctgccc ttcatgggga cagcccagcc aggccccagc aggctaagga gcagccaggg5701 cctgagcgcc ccattccagc tggggatggg aaggtgtgcg tctcctcacc tccagagcct5761 gacgaaactc acgacccgaa gctgcaacat ttggctccag aagagctcca cactgacaga5821 gagagcccca ggcctggccc atccatgtta ccttcggttc ctaagaagga tgctccaaga5881 gtcatggata aagtcacttc agatgagacc agaggtgcgg aaggaacaga aagttcacct5941 gtggcagatg atatcatcca gcccgctgcc cccgcagacc tggaaagccc aaccttagct6001 gcctcttcct accacggtga tgttgttggc caggtctcta cggatctgat agcccagagc6061 atctccccag ctgctgccca tgcgggtctt cctccctcgg ctgcagaaca catagtttcg6121 ccatctgccc cagctggtga cagagtagaa gcttccactc cctcctgccc agatccggcc6181 aaggacctca gcaggagttc cgattctgaa gaggcatttg agaccccgga gtcaacgacc6241 cctgtcaaag ctccgccagc tccaccccca ccaccccccg aagtcatccc agaacccgag6301 gtcagcacac agccaccccc ggaagaacca ggatgtggtt ctgagacagt ccctgtccct6361 gatggcccac ggagcgactc ggtggaagga agtcccttcc gtcccccgtc acactccttc6421 tctgccgtct tcgatgaaga caagccgata gccagcagtg ggacttacaa cttggacttt6481 gacaacattg agcttgtgga tacctttcag accttggagc ctcgtgcctc agacgctaag6541 aatcaggagg gcaaagtgaa cacacggagg aagtccacgg attccgtccc catctctaag6601 tctacactgt cccggtcgct cagcctgcaa gccagtgact ttgatggtgc ttcttcctca6661 ggcaatcccg aggccgtggc ccttgcccca gatgcatata gcacgggttc cagcagtgct6721 tctagtaccc ttaagcgaac taaaaaaccg aggccgcctt ccttaaaaaa gaaacagacc6781 accaagaaac ccacagagac ccccccagtg aaggagacgc aacaggagcc agatgaagag6841 agccttgtcc ccagtgggga gaatctagca tctgagacga aaacggaatc tgccaagacg6901 gaaggtccta gcccagcctt attggaggag acgccccttg agcccgctgt ggggcccaaa6961 gctgcctgcc ctctggactc agagagtgca gaaggggttg tccccccggc ttctggaggt7021 ggcagagtgc agaactcacc ccctgtcggg aggaaaacgc tgcctcttac cacggccccg7081 gaggcagggg aggtaacccc atcggatagc ggggggcaag aggactctcc agccaaaggg7141 ctctccgtaa ggctggagtt tgactattct gaggacaaga gtagttggga caaccagcag7201 gaaaaccccc ctcctaccaa aaagataggc aaaaagccag ttgccaaaat gcccctgagg7261 aggccaaaga tgaaaaagac acccgagaaa cttgacaaca ctcctgcctc acctcccaga7321 tcccctgctg aacccaatga catccccatt gctaaaggta cttacacctt tgatattgac7381 aagtgggatg accccaattt taaccctttt tcttccacct caaaaatgca ggagtctccc7441 aaactgcccc aacaatcata caactttgac ccagacacct gtgatgagtc cgttgacccc7501 tttaagacat cctctaagac ccccagctca ccttctaaat ccccagcctc ctttgagatc7561 ccagccagtg ctatggaagc caatggagtg gacggggatg ggctaaacaa gcccgccaag7621 aagaagaaga cgcccctaaa gactgacaca tttagggtga aaaagtcgcc aaaacggtct7681 cctctctctg atccaccttc ccaggacccc accccagctg ctacaccaga aacaccacca7741 gtgatctctg cggtggtcca cgccacagat gaggaaaagc tggcggtcac caaccagaag7801 tggacgtgca tgacagtgga cctagaggct gacaaacagg actacccgca gccctcggac7861 ctgtccacct ttgtaaacga gaccaaattc agttcaccca ctgaggagtt ggattacaga7921 aactcctatg aaattgaata tatggagaaa attggctcct ccttacctca ggacgacgat7981 gccccgaaga agcaggcctt gtaccttatg tttgacactt ctcaggagag ccctgtcaag8041 tcatctcccg tccgcatgtc agagtccccg acgccgtgtt cagggtcaag ttttgaagag8101 actgaagccc ttgtgaacac tgctgcgaaa aaccagcatc ctgtcccacg aggactggcc8161 cctaaccaag agtcacactt gcaggtgcca gagaaatcct cccagaagga gctggaggcc8221 atgggcttgg gcaccccttc agaagcgatt gaaattacag ctcccgaggg ctcctttgcc8281 tctgctgacg ccctcctcag caggctagct caccccgtct ctctctgtgg tgcacttgac8341 tatctggagc ccgacttagc agaaaagaac cccccactat tcgctcagaa actccaggag8401 gagttagagt ttgccatcat gcggatagaa gccctgaagc tggccaggca gattgctttg8461 gcttcccgca gccaccagga tgccaagaga gaggctgctc acccaacaga cgtctccatc8521 tccaaaacag ccttgtactc ccgcatcggg accgctgagg tggagaaacc tgcaggcctt8581 ctgttccagc agcccgacct ggactctgcc ctccagatcg ccagagcaga gatcataacc8641 aaggagagag aggtctcaga atggaaagat aaatatgaag aaagcaggcg ggaagtgatg8701 gaaatgagga aaatagtggc cgagtatgag aagaccatcg ctcagatgat agaggacgaa8761 cagagagaga agtcagtctc ccaccagacg gtgcagcagc tggttctgga gaaggagcaa8821 gccctggccg acctgaactc cgtggagaag tctctggccg acctcttcag aagatatgag8881 aagatgaagg aggtcctaga aggcttccgc aagaatgaag aggtgttgaa gagatgtgcg8941 caggagtacc tgtcccgggt gaagaaggag gagcagaggt accaggccct gaaggtgcac9001 gcggaggaga aactggacag ggccaatgct gagattgctc aggttcgagg caaggcccag9061 caggagcaag ccgcccacca ggccagcctg cggaaggagc agctgcgagt ggacgccctg9121 gaaaggacgc tggagcagaa gaataaagaa atagaagaac tcaccaagat ttgtgacgaa9181 ctgattgcca aaatggggaa aagctaactc tgaaccgaat gttttggact taactgttgc9241 gtgcaatatg accgtcggca cactgctgtt cctccagttc catggacagg ttctgttttc9301 actttttcgt atgcactact gtatttcctt tctaaataaa attgatttga ttgtatgcag9361 tactaaggag actatcagaa tttcttgcta ttggtttgca ttttcctagt ataattcata9421 gcaagttgac ctcagagttc ctgtatcagg gagattgtct gattctctaa taaaagacac9481 attgctgacc ttggccttgc cctttgtaca caagttccca gggtgagcag cttttggatt9541 taatatgaac atgtacagcg tgcataggga ctcttgcctt aaggagtgta aacttgatct9601 gcatttgctg atttgttttt aaaaaaacaa gaaatgcatg tttcaaataa aattctctat9661 tgtaaataaa attttttctt tggatcttgg caaaaaaaaa aaaaaa

SEQ ID NO: 158 is the amino acid sequence of the FGFR3-TACC3-1 fusionprotein. The bolded text corresponds to the FGFR3 protein:

MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDRVLTVTSTDFKESALRKQSLYLKFDPLLRDSPGRPVPVATETSSMHGANETPSGRPREAKLVEFDFLGALDIPVPGPPPGVPAPGGPPLSTGPIVDLLQYSQKDLDAVVKATQEENRELRSRCEELHGKNLELGKIMDRFEEVVYQAMEEVQKQKELSKAEIQKVLKEKDQLTTDLNSMEKSFSDLFKRFEKQKEVIEGYRKNEESLKKCVEDYLARITQEGQRYQALKAHAEEKLQLANEEIAQVRSKAQAEALALQASLRKEQMRIQSLEKTVEQKTKENEELTRICDDLISKMEKI

SEQ ID NO: 159 is the amino acid sequence of the FGFR3-TACC3-2 fusionprotein. The bolded text corresponds to the FGFR3 protein:

MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDRVLTVTSTDVSAGSGLVPPAYAPPPAVPGHPSGRPREAKLVEFDFLGALDIPVPGPPPGVPAPGGPPLSTGPIVDLLQYSQKDLDAVVKATQEENRELRSRCEELHGKNLELGKIMDRFEEVVYQAMEEVQKQKELSKAEIQKVLKEKDQLTTDLNSMEKSFSDLFKRFEKQKEVIEGYRKNEESLKKCVEDYLARITQEGQRYQALKAHAEEKLQLANEEIAQVRSKAQAEALALQASLRKEQMRIQSLEKTVEQKTKENEE LTRICDDLISKMEKI

SEQ ID NO: 160 is the amino acid sequence of the FGFR3-TACC3-3 fusionprotein. The bolded text corresponds to the FGFR3 protein:

MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDRVLTVTSTDVPGPPPGVPAPGGPPLSTGPIVDLLQYSQKDLDAVVKATQEENRELRSRCEELHGKNLELGKIMDRFEEVVYQAMEEVQKQKELSKAEIQKVLKEKDQLTTDLNSMEKSFSDLFKRFEKQKEVIEGYRKNEESLKKCVEDYLARITQEGQRYQALKAHAEEKLQLANEEIAQVRSKAQAEALALQASLRKEQMRIQSLEKTVEQKTKE NEELTRICDDLISKMEKI

SEQ ID NO: 161 is the amino acid sequence of the FGFR3-TACC3-4 fusionprotein. The bolded text corresponds to the FGFR3 protein:

MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDRVLTVTSTDVKATQEENRELRSRCEELHGKNLELGKIMDRFEEVVYQAMEEVQKQKELSKAEIQKVLKEKDQLTTDLNSMEKSFSDLFKRFEKQKEVIEGYRKNEESLKKCVEDYLARITQEGQRYQALKAHAEEKLQLANEEIAQVRSKAQAEALALQASLRKEQMRIQSLEKTVEQKTKENEELTRICDDLISKMEKI

SEQ ID NO: 539 is the amino acid sequence of FGFR3ex17-TACC3ex11. Thesequence corresponding to FGFR3 is underlined. The sequencecorresponding to TACC3 is shaded:

MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGESHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCT

SEQ ID NO: 540 is the amino acid sequence of FGFR3ex17-TACC3ex8. Thesequence corresponding to FGFR3 is underlined. The sequencecorresponding to TACC3 is shaded:

MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCT

SEQ ID NO: 541 is the amino acid sequence of FGFR3ex17-TACC3ex10. Thesequence corresponding to FGFR3 is underlined. The sequencecorresponding to TACC3 is shaded:

MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPVEELFKLLKEGHRMD

SEQ ID NO: 542 is the amino acid sequence of FGFR3ex17-TACC3ex6. Thesequence corresponding to FGFR3 is underlined. The sequencecorresponding to TACC3 is shaded:

MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDR

SEQ ID NO: 543 is the amino acid sequence of FGFR3ex18-TACC3ex13. Thesequence corresponding to FGFR3 is underlined. The sequencecorresponding to TACC3 is shaded:

MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDR

SEQ ID NO: 544 is the amino acid sequence of FGFR3ex18-TACC3ex9_INS66BP.The sequence corresponding to FGFR3 is underlined. The sequencecorresponding to TACC3 is shaded. The sequence corresponding the the 66bp intronic insert is double underlined:

MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDR

SEQ ID NO: 545 is the amino acid sequence of FGFR3ex18-TACC3ex5. Thesequence corresponding to FGFR3 is underlined. The sequencecorresponding to TACC3 is shaded:

MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAP

SEQ ID NO: 546 is the amino acid sequence of FGFR3ex18-TACC3ex5_INS33bp. The sequence corresponding to FGFR3 is underlined. The sequencecorresponding to TACC3 is shaded. The sequence corresponding the the 33bp intronic insert is double underlined:

MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDR

SEQ ID NO: 547 is the amino acid sequence of FGFR3ex18-TACC3ex4. Thesequence corresponding to FGFR3 is underlined. The sequencecorresponding to TACC3 is shaded.

MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRSPPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDR

The Genbank ID for the FGFR4 gene is 2264. Three isoforms are listed forFGFR4, e.g., having Genebank Accession Nos. NP_(—)002002 (correspondingnucleotide sequence NM_(—)002011); NP_(—)075252 (correspondingnucleotide sequence NM_(—)022963); NP_(—)998812 (correspondingnucleotide sequence NM_(—)213647).

As used herein, a “FGFR fusion molecule” can be a nucleic acid (e.g.,synthetic, purified, and/or recombinant) which encodes a polypeptidecorresponding to a fusion protein comprising a tyrosine kinase domain ofan FGFR protein fused to a polypeptide that constitutively activates thetyrosine kinase domain of the FGFR protein, or a nucleic acid encoding afusion protein comprising a transforming acidic coiled-coil (TACC)domain fused to a polypeptide with a tyrosine kinase domain, wherein theTACC domain constitutively activates the tyrosine kinase domain. It canalso be a fusion protein comprising a tyrosine kinase domain of an FGFRprotein fused to a polypeptide that constitutively activates thetyrosine kinase domain of the FGFR protein, or a fusion proteincomprising a transforming acidic coiled-coil (TACC) domain fused to apolypeptide with a tyrosine kinase domain, wherein the TACC domainconstitutively activates the tyrosine kinase domain. For example, a FGFRfusion molecule can include a FGFR1-TACC1 (e.g., comprising the aminoacid sequence shown in SEQ ID NO: 150, or comprising the nucleic acidsequence shown in SEQ ID NO: 88), FGFR2-TACC2, FGFR3-TACC3 (e.g.,comprising the amino acid sequence shown in SEQ ID NOS: 79, 158-161, or539-547 or comprising the nucleic acid sequence shown in SEQ ID NOS:80-82, 84, 94-145, 515, 517, 519-527, or 530-538), or other FGFR-TACCfusion proteins (e.g., an N-terminal fragment of FGFR4 containing itstyrosine kinase domain fused to a fragment of TACC1, TACC2, or TACC3).For example, a FGFR fusion molecule can include a FGFR1-containingfusion comprising the amino acid sequence corresponding to GenebankAccession no. NP_(—)001167534, NP_(—)001167535, NP_(—)001167536,NP_(—)001167537, NP_(—)001167538, NP_(—)056934, NP_(—)075593,NP_(—)075594, or NP_(—)075598; or a FGFR1-containing fusion comprisingthe nucleotide sequence corresponding to Genebank Accession no.NM_(—)001174063, NM_(—)001174064, NM_(—)001174065, NM_(—)001174066,NM_(—)001174067, NM_(—)015850, NM_(—)023105, NM_(—)023106, orNM_(—)023110. For example, a FGFR fusion molecule can include aFGFR2-containing fusion comprising the amino acid sequence correspondingto Genebank Accession no. NP_(—)000132, NP_(—)001138385,NP_(—)001138386, NP_(—)001138387, NP_(—)001138388, NP_(—)001138389,NP_(—)001138390, NP_(—)001138391, or NP_(—)075259; or a FGFR2-containingfusion comprising the nucleotide sequence corresponding to GenebankAccession no. NM_(—)000141, NM_(—)001144913, NM_(—)001144914,NM_(—)001144915, NM_(—)001144916, NM_(—)001144917, NM_(—)001144918,NM_(—)001144919, or NM_(—)022970. For example, a FGFR fusion moleculecan include a FGFR3-containing fusion comprising the amino acid sequencecorresponding to Genebank Accession no. NP_(—)000133, NP_(—)001156685,or NP_(—)075254; or a FGFR3-containing fusion comprising the nucleotidesequence corresponding to Genebank Accession no. NM_(—)000142,NM_(—)001163213, or NM_(—)022965. For example, a FGFR fusion moleculecan include a FGFR4-containing fusion comprising the amino acid sequencecorresponding to Genebank Accession no. NP_(—)002002, NP_(—)075252, orNP_(—)998812; or a FGFR4-containing fusion comprising the nucleotidesequence corresponding to Genebank Accession no. NM_(—)002011,NM_(—)022963, or NM_(—)213647. A FGFR fusion molecule can also include atyrosine kinase domain of an FGFR protein fused to a protein encoded byany one of the genes listed in Table 7. A FGFR fusion molecule caninclude a variant of the above described examples, such as a fragmentthereof. Table 7. Fusion Partners

gene gene gene gene ABCA13 C21orf29 CAMKK1 DNAJC6 ABCC1 CACNA1C CAMSAP1DYRK3 ABCC12 CACNA1G CAMTA1 EIF2C2 ABCC6 CNTNAP4 CAP2 FAM184B ABL1 CUL3CCDC147 FREM2 ADAM12 DMD CCDC158 GDPD2 ADCY10 DUSP27 CELF2 GLI3 ADCY2ECE1 CILP IL1RN ADCY8 EYS CMYA5 ISX AGBL4 FAM172A COL14A1 KIDINS220AHNAK FAM184B CORO7 LRBA ANXA7 FGFR4 CSMD2 LY75 AP4S1 ITGAV CUL3 MDH2AQP2 LRP1 DDI2 MMP12 ARMC6 LY75 DEPDC5 N4BP2L2 ATP5B MAPKAP1 DEPDC7 NCF2ATP6AP1L MYT1 DI10L NCOR1 ATP6V0D2 NCF2 DMD NCRNA00157 ATXN1 NCOR1 EDANRXN3 BAHD1 NHSL2 EFHC1 PARP16 BBX NKAIN2 EFS PLA2G2F BCA10 NR3C1 EIF2C2PLEK2 C15orf23 NUP188 ENTPD2 PRKCH C15orf33 OSBPL10 EYS PTPRS C21orf29PACSIN1 FAM160A1 ROBO1 C2CD3 PARP16 MUSK SASH3 C6orf170 PDZRN4 NEUROG1SH3BP5 C7orf44 POLM NHSL2 SLC44A2 CACNA1C PPP1R3A NR3C1 SLC5A4 CACNA1GPSEN1 ODZ1 SNX5 FAM168A PTPRD PCDH12 SORCS2 FAM172A PTPRS PLCL1 SRRM1FAM192A RALYL PLEKHM3 SSX3 FAM19A2 RERE PLOD3 STAG2 FBXL4 RIMBP2 PRKCHSTK24 FH RNF216 PSEN1 SURF6 FREM2 SDAD1 SEPT5 SYNPO2 GAPVD1 SEC14L3SLC44A2 TAF1 GLI3 SH3RF3 SNTA1 TMEM80 GPR182 SLC9A1 USP48 TNFRSF10BGSTA3 SMOC2 VSNL1 TTYH1 IGFBP3 SNX5 WDFY1 UNC93B1 ITGA9 TACC2 WISP2VSNL1 ITGB2 SRGAP1 XRRA1 XRCC4 JOSD2 SSX3 LRRC4B ZNF410 KIDINS220 SUMF1LRRK2 TRIOBP LAMA2 SYNPO2 MAPKAP1 TTYH1 LCLAT1 TNFRSF10B MST1R LRBA LIN9

For example, a FGFR fusion molecule can include a FGFR3-containingfusion comprising the amino acid sequence corresponding to residues1-760 of FGFR3 (e.g. SEQ ID NO: 90) fused to the amino acid sequencecorresponding residues 648-838 of TACC3 (e.g. SEQ ID NO: 92). A FGFRfusion molecule can also include a FGFR3-containing fusion comprisingthe amino acid sequence corresponding to residues 1-760 of FGFR3 (e.g.SEQ ID NO: 90) fused to the amino acid sequence corresponding residues549-838 of TACC3 (e.g. SEQ ID NO: 92). A FGFR fusion molecule caninclude a FGFR3-containing fusion comprising the amino acid sequencecorresponding to residues 1-760 of FGFR3 (e.g. SEQ ID NO: 90) fused tothe amino acid sequence corresponding residues 613-838 of TACC3 (e.g.SEQ ID NO: 92). A FGFR fusion molecule can include a FGFR3-containingfusion comprising the amino acid sequence corresponding to residues1-760 of FGFR3 (e.g. SEQ ID NO: 90) fused to the amino acid sequencecorresponding residues 488-838 of TACC3 (e.g. SEQ ID NO: 92). A FGFRfusion molecule can include a FGFR3-containing fusion comprising theamino acid sequence corresponding to residues 1-781 of FGFR3 (e.g. SEQID NO: 90) fused to the amino acid sequence corresponding residues689-838 of TACC3 (e.g. SEQ ID NO: 92). A FGFR fusion molecule caninclude a FGFR3-containing fusion comprising the amino acid sequencecorresponding to residues 1-765 of FGFR3 (e.g. SEQ ID NO: 90) fused tothe amino acid sequence corresponding residues 583-838 of TACC3 (e.g.SEQ ID NO: 92). A FGFR fusion molecule can include a FGFR3-containingfusion comprising the amino acid sequence corresponding to residues1-767 of FGFR3 (e.g. SEQ ID NO: 90) fused to the amino acid sequencecorresponding residues 462-838 of TACC3 (e.g. SEQ ID NO: 92). A FGFRfusion molecule can include a FGFR3-containing fusion comprising theamino acid sequence corresponding to residues 1-767 of FGFR3 (e.g. SEQID NO: 90) fused to the amino acid sequence corresponding residues472-838 of TACC3 (e.g. SEQ ID NO: 92). A FGFR fusion molecule caninclude a FGFR3-containing fusion comprising the amino acid sequencecorresponding to residues 1-780 of FGFR3 (e.g. SEQ ID NO: 90) fused tothe amino acid sequence corresponding residues 432-838 of TACC3 (e.g.SEQ ID NO: 92). A FGFR fusion molecule can include a FGFR1-containingfusion comprising the amino acid sequence corresponding to residues1-762 of FGFR1 (e.g. SEQ ID NOS: 146, 185) fused to the amino acidsequence corresponding residues 571-805 of TACC1 (e.g. SEQ ID NO: 148).For example, a FGFR fusion molecule can include SEQ ID NOs: 539-543,545, and 547.

The alteration in a chromosome region occupied by a FGFR fusionmolecule, e.g., a FGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3 or otherFGFR-TACC nucleic acid, can result in amino acid substitutions, RNAsplicing or processing, product instability, the creation of stopcodons, production of oncogenic fusion proteins, frame-shift mutations,and/or truncated polypeptide production. A FGFR fusion molecule caninclude FGFR and TACC exons joined in the fused mRNA or cDNA. A FGFRfusion molecule can also include FGFR and TACC exons joined in the fusedmRNA or cDNA along with the presence of FGFR or TACC introns that arespliced in the fusion cDNA. FGFR or TACC introns can encode amino acidsof the FGFR fusion molecule. For example, a FGFR fusion molecule caninclude a FGFR3-containing fusion comprising the amino acid sequencecorresponding to residues 1-765 of FGFR3 (e.g. SEQ ID NO: 90) fused to a22 amino acid sequence encoded by a TACC3 intron fused to the amino acidsequence corresponding to residues 583-838 of TACC3 (e.g. SEQ ID NO:92). For example, a FGFR fusion molecule can include a FGFR3-containingfusion comprising the amino acid sequence corresponding to residues1-767 of FGFR3 (e.g. SEQ ID NO: 90) fused to a 11 amino acid sequenceencoded by a TACC3 intron fused to the amino acid sequence correspondingto residues 472-838 of TACC3 (e.g. SEQ ID NO: 92). For example, a FGFRfusion molecule can include SEQ ID NOs: 544 and 546.

A FGFR fusion protein can also include a fusion protein encoded by anFGFR3-TACC3 nucleic acid, wherein FGFR3-TACC3 comprises a combination ofintrons and exons 1-18 of FGFR3 located on human chromosome 4p16 spliced5′ to a combination of introns and exons 4-16 of TACC3 located on humanchromosome 4p16, wherein a genomic breakpoint occurs in any one ofintrons or exons 1-18 of FGFR3 and any one of introns or exons 3-16 ofTACC3. For example, a FGFR fusion protein can also include a fusionprotein encoded by an FGFR3-TACC3 nucleic acid, wherein the nucleic acidcomprises exons 1-17 of FGFR3 located on human chromosome 4p16 spliced5′ to exons 11-16 of TACC3 located on human chromosome 4p16. In oneembodiment, a genomic breakpoint occurs in intron 17 of FGFR3 and inintron 10 of TACC3. For example, a FGFR fusion protein can also includea fusion protein encoded by an FGFR3-TACC3 nucleic acid, wherein thenucleic acid comprises exons 1-17 of FGFR3 located on human chromosome4p16 spliced 5′ to exons 8-16 of TACC3 located on human chromosome 4p16.In one embodiment, a genomic breakpoint occurs in intron 17 of FGFR3 andin intron 7 of TACC3. For example, a FGFR fusion protein can alsoinclude a fusion protein encoded by an FGFR3-TACC3 nucleic acid, whereinthe nucleic acid comprises exons 1-17 of FGFR3 located on humanchromosome 4p16 spliced 5′ to exons 10-16 of TACC3 located on humanchromosome 4p16. In one embodiment, a genomic breakpoint occurs in exon18 of FGFR3 and in intron 9 of TACC3. For example, a FGFR fusion proteincan also include a fusion protein encoded by an FGFR3-TACC3 nucleicacid, wherein the nucleic acid comprises exons 1-17 of FGFR3 located onhuman chromosome 4p16 spliced 5′ to exons 6-16 of TACC3 located on humanchromosome 4p16. In one embodiment, a genomic breakpoint occurs inintron 17 of FGFR3 and in intron 5 of TACC3. In one embodiment, agenomic breakpoint occurs in intron 17 of FGFR3 and in exon 5 of TACC3.For example, a FGFR fusion protein can also include a fusion proteinencoded by an FGFR3-TACC3 nucleic acid, wherein the nucleic acidcomprises exons 1-18 of FGFR3 located on human chromosome 4p16 spliced5′ to exons 13-16 of TACC3 located on human chromosome 4p16. In oneembodiment, a genomic breakpoint occurs in exon 18 of FGFR3 and inintron 12 or exon 13 of TACC3. For example, a FGFR fusion protein canalso include a fusion protein encoded by an FGFR3-TACC3 nucleic acid,wherein the nucleic acid comprises exons 1-18 of FGFR3 located on humanchromosome 4p16 spliced 5′ to a portion of intron 8 of TACC3 and exons9-16 of TACC3 located on human chromosome 4p16. In one embodiment, agenomic breakpoint occurs in exon 18 of FGFR3 and in intron 8 of TACC3.For example, a FGFR fusion protein can also include a fusion proteinencoded by an FGFR3-TACC3 nucleic acid, wherein the nucleic acidcomprises exons 1-18 of FGFR3 located on human chromosome 4p16 spliced5′ to exons 5-16 of TACC3 located on human chromosome 4p16. In oneembodiment, a genomic breakpoint occurs in exon 18 of FGFR3 and inintron 4 or exon 5 of TACC3. For example, a FGFR fusion protein can alsoinclude a fusion protein encoded by an FGFR3-TACC3 nucleic acid, whereinthe nucleic acid comprises exons 1-18 of FGFR3 located on humanchromosome 4p16 spliced 5′ to a portion of intron 4 of TACC 3 and exons5-16 of TACC3 located on human chromosome 4p16. In one embodiment, agenomic breakpoint occurs in exon 18 of FGFR3 and in intron 4 or exon 5of TACC3. For example, a FGFR fusion protein can also include a fusionprotein encoded by an FGFR3-TACC3 nucleic acid, wherein the nucleic acidcomprises exons 1-18 of FGFR3 located on human chromosome 4p16 spliced5′ to exons 4-16 of TACC3 located on human chromosome 4p16. In oneembodiment, a genomic breakpoint occurs in exon 18 of FGFR3 and inintron 3 or exon 4 of TACC3. For example, a FGFR fusion protein can alsoinclude a fusion protein encoded by an FGFR3-TACC3 nucleic acid, whereinthe nucleic acid comprises exons 1-17 of FGFR3 and a portion of intron17 of FGFR3 located on human chromosome 4p16 spliced 5′ to exons 9-16 ofTACC3 located on human chromosome 4p16. In one embodiment, a genomicbreakpoint occurs in intron 17 of FGFR3 and in exon 9 of TACC3. Forexample, a FGFR fusion protein can also include a fusion protein encodedby an FGFR1-TACC1 nucleic acid, wherein the nucleic acid comprises exons1-17 of FGFR1 located on human chromosome 8p11 spliced 5′ to exons 7-13of TACC1 located on human chromosome 8p11. In one embodiment, a genomicbreakpoint occurs in intron 17 or exon 17 of FGFR1 and in intron 6 orexon 7 of TACC1.

In one embodiment, the coordinates comprising FGFR3 translocationscomprise chr4:1,795,039-1,810,599. In a further embodiment, the genomicbreakpoint coordinate according to the genome build GRCh37/hg19 forFGFR3 is chr4:1,808,808, chr4:1,808,843, chr4:1,809,083, chr4:1,808,785,chr4:1,808,700, chr4:1,808,864, chr4:1,808,678, chr4:1, 808,798, orchr4:1,808,723. In a further embodiment, the coordinates comprisingTACC3 translocations comprise chr4:1,723,217-1,746,905. In a furtherembodiment, the genomic breakpoint coordinate according to the genomebuild GRCh37/hg19 for TACC3 is chr4:1,732,648, chr4:1,732,757,chr4:1,739,187, chr4:1,737,091, chr4:1,737,062, chr4:1,737741,chr4:1,739,662, chr4:1,739,600, or chr4:1,738,989.

The nucleic acid can be any type of nucleic acid, including genomic DNA,complementary DNA (cDNA), recombinant DNA, synthetic or semi-syntheticDNA, as well as any form of corresponding RNA. A cDNA is a form of DNAartificially synthesized from a messenger RNA template and is used toproduce gene clones. A synthetic DNA is free of modifications that canbe found in cellular nucleic acids, including, but not limited to,histones and methylation. For example, a nucleic acid encoding a FGFRfusion molecule can comprise a recombinant nucleic acid encoding such aprotein. The nucleic acid can be a non-naturally occurring nucleic acidcreated artificially (such as by assembling, cutting, ligating oramplifying sequences). It can be double-stranded or single-stranded.

The invention further provides for nucleic acids that are complementaryto a FGFR fusion molecule. Complementary nucleic acids can hybridize tothe nucleic acid sequence described above under stringent hybridizationconditions. Non-limiting examples of stringent hybridization conditionsinclude temperatures above 30° C., above 35° C., in excess of 42° C.,and/or salinity of less than about 500 mM, or less than 200 mM.Hybridization conditions can be adjusted by the skilled artisan viamodifying the temperature, salinity and/or the concentration of otherreagents such as SDS or SSC.

According to the invention, protein variants can include amino acidsequence modifications. For example, amino acid sequence modificationsfall into one or more of three classes: substitutional, insertional ordeletional variants. Insertions can include amino and/or carboxylterminal fusions as well as intrasequence insertions of single ormultiple amino acid residues. Insertions ordinarily will be smallerinsertions than those of amino or carboxyl terminal fusions, forexample, on the order of one to four residues. Deletions arecharacterized by the removal of one or more amino acid residues from theprotein sequence. These variants ordinarily are prepared bysite-specific mutagenesis of nucleotides in the DNA encoding theprotein, thereby producing DNA encoding the variant, and thereafterexpressing the DNA in recombinant cell culture.

In one embodiment, a FGFR fusion molecule comprises a protein orpolypeptide encoded by a nucleic acid sequence encoding a FGFR fusionmolecule, such as the sequences shown in SEQ ID NOS: 80-82, 84, 94-145,515, 517, 519-527, or 530-538. In some embodiments, the nucleic acidsequence encoding a FGFR fusion molecule is about 70%, about 75%, about80%, about 85%, about 90%, about 93%, about 95%, about 97%, about 98%,or about 99% identical to SEQ ID NOS: 80-82, 84, 94-145, 515, 517,519-527, or 530-538. In another embodiment, the polypeptide can bemodified, such as by glycosylations and/or acetylations and/or chemicalreaction or coupling, and can contain one or several non-natural orsynthetic amino acids. An example of a FGFR fusion molecule is thepolypeptide having the amino acid sequence shown in SEQ ID NOS: 79, 88,150, 158-161, or 539-547. In some embodiments, the FGFR fusion moleculethat is a polypeptide is about 70%, about 75%, about 80%, about 85%,about 90%, about 93%, about 95%, about 97%, about 98%, or about 99%identical to SEQ ID NOS: 79, 88, 150, 158-161, or 539-547. In anotherembodiment, a FGFR fusion molecule can be a fragment of a FGFR fusionprotein. For example, the FGFR fusion molecule can encompass any portionof at least about 8 consecutive amino acids of SEQ ID NOS: 79, 88, 150,158-161, or 539-547. The fragment can comprise at least about 10 aminoacids, a least about 20 amino acids, at least about 30 amino acids, atleast about 40 amino acids, a least about 50 amino acids, at least about60 amino acids, or at least about 75 amino acids of SEQ ID NOS: 79, 88,150, 158-161, or 539-547. Fragments include all possible amino acidlengths between about 8 and 100 about amino acids, for example, lengthsbetween about 10 and 100 amino acids, between about 15 and 100 aminoacids, between about 20 and 100 amino acids, between about 35 and 100amino acids, between about 40 and 100 amino acids, between about 50 and100 amino acids, between about 70 and 100 amino acids, between about 75and 100 amino acids, or between about 80 and 100 amino acids. Fragmentsinclude all possible amino acid lengths between about 100 and 800 aminoacids, for example, lengths between about 125 and 800 amino acids,between about 150 and 800 amino acids, between about 175 and 800 aminoacids, between about 200 and 800 amino acids, between about 225 and 800amino acids, between about 250 and 800 amino acids, between about 275and 800 amino acids, between about 300 and 800 amino acids, betweenabout 325 and 800 amino acids, between about 350 and 800 amino acids,between about 375 and 800 amino acids, between about 400 and 800 aminoacids, between about 425 and 800 amino acids, between about 450 and 800amino acids, between about 475 and 800 amino acids, between about 500and 800 amino acids, between about 525 and 800 amino acids, betweenabout 550 and 800 amino acids, between about 575 and 800 amino acids,between about 600 and 800 amino acids, between about 625 and 800 aminoacids, between about 650 and 800 amino acids, between about 675 and 800amino acids, between about 700 and 800 amino acids, between about 725and 800 amino acids, between about 750 and 800 amino acids, or betweenabout 775 and 800 amino acids.

Chemical Synthesis.

Nucleic acid sequences encoding a FGFR fusion molecule can besynthesized, in whole or in part, using chemical methods known in theart. Alternatively, a polypeptide can be produced using chemical methodsto synthesize its amino acid sequence, such as by direct peptidesynthesis using solid-phase techniques. Protein synthesis can either beperformed using manual techniques or by automation. Automated synthesiscan be achieved, for example, using Applied Biosystems 431A PeptideSynthesizer (Perkin Elmer).

Optionally, polypeptides fragments can be separately synthesized andcombined using chemical methods to produce a full-length molecule. Forexample, these methods can be utilized to synthesize a fusion protein ofthe invention. In one embodiment, the fusion protein comprises atyrosine kinase domain of an FGFR protein fused to a polypeptide thatconstitutively activates the tyrosine kinase domain of the FGFR protein.In another embodiment, a fusion protein comprises a transforming acidiccoiled-coil (TACC) domain fused to a polypeptide with a tyrosine kinasedomain, wherein the TACC domain constitutively activates the tyrosinekinase domain. An example of a fusion protein is the FGFR1-TACC1polypeptide, which comprises the amino acid sequence shown in SEQ ID NO:150. An example of a fusion protein is the FGFR3-TACC3 polypeptide,which has the amino acid sequence comprising SEQ ID NO: 79, 158, 159,160, 161, 539, 540, 541, 542, 543, 544, 545, 546, or 547.

Obtaining, Purifying and Detecting FGFR Fusion Molecules.

A polypeptide encoded by a nucleic acid, such as a nucleic acid encodinga FGFR fusion molecule, or a variant thereof, can be obtained bypurification from human cells expressing a protein or polypeptideencoded by such a nucleic acid. Non-limiting purification methodsinclude size exclusion chromatography, ammonium sulfate fractionation,ion exchange chromatography, affinity chromatography, and preparativegel electrophoresis.

A synthetic polypeptide can be substantially purified via highperformance liquid chromatography (HPLC), such as ion exchangechromatography (IEX-HPLC). The composition of a synthetic polypeptide,such as a FGFR fusion molecule, can be confirmed by amino acid analysisor sequencing.

Other constructions can also be used to join a nucleic acid sequenceencoding a polypeptide/protein of the claimed invention to a nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). Including cleavablelinker sequences (i.e., those specific for Factor Xa or enterokinase(Invitrogen, San Diego, Calif.)) between the purification domain and apolypeptide encoded by a nucleic acid of the invention also can be usedto facilitate purification. For example, the skilled artisan can use anexpression vector encoding 6 histidine residues that precede athioredoxin or an enterokinase cleavage site in conjunction with anucleic acid of interest. The histidine residues facilitate purificationby immobilized metal ion affinity chromatography, while the enterokinasecleavage site provides a means for purifying the polypeptide encoded by,for example, an FGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3, other FGFR-TACC,FGFR-containing, or TACC containing nucleic acid.

Host cells which contain a nucleic acid encoding a FGFR fusion molecule,and which subsequently express the same, can be identified by variousprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip-based technologies for the detection and/or quantification ofnucleic acid or protein. For example, the presence of a nucleic acidencoding a FGFR fusion molecule can be detected by DNA-DNA or DNA-RNAhybridization or amplification using probes or fragments of nucleicacids encoding the same. In one embodiment, a nucleic acid fragment of aFGFR fusion molecule can encompass any portion of at least about 8consecutive nucleotides of SEQ ID NOS: 80-82, 84, 94-145, 515, 517,519-527, or 530-538. In another embodiment, the fragment can comprise atleast about 10 consecutive nucleotides, at least about 15 consecutivenucleotides, at least about 20 consecutive nucleotides, or at leastabout 30 consecutive nucleotides of SEQ ID NOS: 80-82, 84, 94-145, 515,517, 519-527, 530-538. Fragments can include all possible nucleotidelengths between about 8 and about 100 nucleotides, for example, lengthsbetween about 15 and about 100 nucleotides, or between about 20 andabout 100 nucleotides. Nucleic acid amplification-based assays involvethe use of oligonucleotides selected from sequences encoding a FGFRfusion molecule nucleic acid, or FGFR fusion molecule nucleic acid todetect transformants which contain a nucleic acid encoding a protein orpolypeptide of the same.

Protocols are known in the art for detecting and measuring theexpression of a polypeptide encoded by a nucleic acid, such as a nucleicacid encoding a FGFR fusion molecule, using either polyclonal ormonoclonal antibodies specific for the polypeptide. Non-limitingexamples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), immunostaining, and fluorescence activated cellsorting (FACS). A two-site, monoclonal-based immunoassay usingmonoclonal antibodies reactive to two non-interfering epitopes on apolypeptide encoded by a nucleic acid, such as a nucleic acid encoding aFGFR fusion molecule, can be used, or a competitive binding assay can beemployed.

Labeling and conjugation techniques are known by those skilled in theart and can be used in various nucleic acid and amino acid assays.Methods for producing labeled hybridization or PCR probes for detectingsequences related to nucleic acid sequences encoding a protein, such asFGFR fusion molecule, include, but are not limited to, oligolabeling,nick translation, end-labeling, or PCR amplification using a labelednucleotide. Alternatively, nucleic acid sequences, such as nucleic acidsencoding a FGFR fusion molecule, can be cloned into a vector for theproduction of an mRNA probe. Such vectors are known in the art, arecommercially available, and can be used to synthesize RNA probes invitro by addition of labeled nucleotides and an appropriate RNApolymerase such as T7, T3, or SP6. These procedures can be conductedusing a variety of commercially available kits (Amersham PharmaciaBiotech, Promega, and US Biochemical). Suitable reporter molecules orlabels which can be used for ease of detection include radionuclides,enzymes, and fluorescent, chemiluminescent, or chromogenic agents, aswell as substrates, cofactors, inhibitors, and/or magnetic particles.

A fragment can be a fragment of a protein, such as a FGFR fusionprotein. For example, a fragment of a FGFR fusion molecule can encompassany portion of at least about 8 consecutive amino acids of SEQ ID NOS:79, 88, 150, 158-161, or 539-547. The fragment can comprise at leastabout 10 consecutive amino acids, at least about 20 consecutive aminoacids, at least about 30 consecutive amino acids, at least about 40consecutive amino acids, a least about 50 consecutive amino acids, atleast about 60 consecutive amino acids, at least about 70 consecutiveamino acids, at least about 75 consecutive amino acids, at least about80 consecutive amino acids, at least about 85 consecutive amino acids,at least about 90 consecutive amino acids, at least about 95 consecutiveamino acids, at least about 100 consecutive amino acids, at least about200 consecutive amino acids, at least about 300 consecutive amino acids,at least about 400 consecutive amino acids, at least about 500consecutive amino acids, at least about 600 consecutive amino acids, atleast about 700 consecutive amino acids, or at least about 800consecutive amino acids of SEQ ID NOS: 79, 88, 150, 158-161, or 539-547.Fragments include all possible amino acid lengths between about 8 and100 about amino acids, for example, lengths between about 10 and about100 amino acids, between about 15 and about 100 amino acids, betweenabout 20 and about 100 amino acids, between about 35 and about 100 aminoacids, between about 40 and about 100 amino acids, between about 50 andabout 100 amino acids, between about 70 and about 100 amino acids,between about 75 and about 100 amino acids, or between about 80 andabout 100 amino acids.

Cell Transfection

Host cells transformed with a nucleic acid sequence of interest can becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The polypeptide produced by a transformedcell can be secreted or contained intracellularly depending on thesequence and/or the vector used. Expression vectors containing a nucleicacid sequence, such as a nucleic acid encoding a FGFR fusion molecule,can be designed to contain signal sequences which direct secretion ofsoluble polypeptide molecules encoded by the nucleic acid. Celltransfection and culturing methods are described in more detail below.

A eukaryotic expression vector can be used to transfect cells in orderto produce proteins encoded by nucleotide sequences of the vector, e.g.those encoding a FGFR fusion molecule. Mammalian cells can contain anexpression vector (for example, one that contains a nucleic acidencoding a fusion protein comprising a tyrosine kinase domain of an FGFRprotein fused to a polypeptide that constitutively activates thetyrosine kinase domain of the FGFR protein, or a nucleic acid encoding afusion protein comprises a transforming acidic coiled-coil (TACC) domainfused to a polypeptide with a tyrosine kinase domain, wherein the TACCdomain constitutively activates the tyrosine kinase domain) viaintroducing the expression vector into an appropriate host cell viamethods known in the art.

A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressedpolypeptide encoded by a nucleic acid, in the desired fashion. Suchmodifications of the polypeptide include, but are not limited to,acetylation, carboxylation, glycosylation, phosphorylation, lipidation,and acylation. Post-translational processing which cleaves a “prepro”form of the polypeptide also can be used to facilitate correctinsertion, folding and/or function. Different host cells which havespecific cellular machinery and characteristic mechanisms forpost-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38),are available from the American Type Culture Collection (ATCC; 10801University Boulevard, Manassas, Va. 20110-2209) and can be chosen toensure the correct modification and processing of the foreign protein.

An exogenous nucleic acid can be introduced into a cell via a variety oftechniques known in the art, such as lipofection, microinjection,calcium phosphate or calcium chloride precipitation,DEAE-dextran-mediated transfection, or electroporation. Electroporationis carried out at approximate voltage and capacitance to result in entryof the DNA construct(s) into cells of interest (such as glioma cells(cell line SF188), neuroblastoma cells (cell lines IMR-32, SK-N-SH, SH-Fand SH-N), astrocytes and the like). Other transfection methods alsoinclude modified calcium phosphate precipitation, polybreneprecipitation, liposome fusion, and receptor-mediated gene delivery.

Cells that will be genetically engineered can be primary and secondarycells obtained from various tissues, and include cell types which can bemaintained and propagated in culture. Non-limiting examples of primaryand secondary cells include epithelial cells, neural cells, endothelialcells, glial cells, fibroblasts, muscle cells (such as myoblasts)keratinocytes, formed elements of the blood (e.g., lymphocytes, bonemarrow cells), and precursors of these somatic cell types.

Vertebrate tissue can be obtained by methods known to one skilled in theart, such a punch biopsy or other surgical methods of obtaining a tissuesource of the primary cell type of interest. In one embodiment, a punchbiopsy or removal (e.g., by aspiration) can be used to obtain a sourceof cancer cells (for example, glioma cells, neuroblastoma cells, and thelike). A mixture of primary cells can be obtained from the tissue, usingmethods readily practiced in the art, such as explanting or enzymaticdigestion (for examples using enzymes such as pronase, trypsin,collagenase, elastase dispase, and chymotrypsin). Biopsy methods havealso been described in U.S. Pat. No. 7,419,661 and PCT applicationpublication WO 2001/32840, and each are hereby incorporated byreference.

Primary cells can be acquired from the individual to whom thegenetically engineered primary or secondary cells are administered.However, primary cells can also be obtained from a donor, other than therecipient, of the same species. The cells can also be obtained fromanother species (for example, rabbit, cat, mouse, rat, sheep, goat, dog,horse, cow, bird, or pig). Primary cells can also include cells from apurified vertebrate tissue source grown attached to a tissue culturesubstrate (for example, flask or dish) or grown in a suspension; cellspresent in an explant derived from tissue; both of the aforementionedcell types plated for the first time; and cell culture suspensionsderived from these plated cells. Secondary cells can be plated primarycells that are removed from the culture substrate and replated, orpassaged, in addition to cells from the subsequent passages. Secondarycells can be passaged one or more times. These primary or secondarycells can contain expression vectors having a gene that encodes a FGFRfusion molecule.

Cell Culturing

Various culturing parameters can be used with respect to the host cellbeing cultured. Appropriate culture conditions for mammalian cells arewell known in the art (Cleveland W L, et al., J Immunol Methods, 1983,56(2): 221-234) or can be determined by the skilled artisan (see, forexample, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D.and Hames, B. D., eds. (Oxford University Press: New York, 1992)). Cellculturing conditions can vary according to the type of host cellselected. Commercially available medium can be utilized. Non-limitingexamples of medium include, for example, Minimal Essential Medium (MEM,Sigma, St. Louis, Mo.); Dulbecco's Modified Eagles Medium (DMEM, Sigma);Ham's F10 Medium (Sigma); HyClone cell culture medium (HyClone, Logan,Utah); RPMI-1640 Medium (Sigma); and chemically-defined (CD) media,which are formulated for various cell types, e.g., CD-CHO Medium(Invitrogen, Carlsbad, Calif.).

The cell culture media can be supplemented as necessary withsupplementary components or ingredients, including optional components,in appropriate concentrations or amounts, as necessary or desired. Cellculture medium solutions provide at least one component from one or moreof the following categories: (1) an energy source, usually in the formof a carbohydrate such as glucose; (2) all essential amino acids, andusually the basic set of twenty amino acids plus cysteine; (3) vitaminsand/or other organic compounds required at low concentrations; (4) freefatty acids or lipids, for example linoleic acid; and (5) traceelements, where trace elements are defined as inorganic compounds ornaturally occurring elements that can be required at very lowconcentrations, usually in the micromolar range.

The medium also can be supplemented electively with one or morecomponents from any of the following categories: (1) salts, for example,magnesium, calcium, and phosphate; (2) hormones and other growth factorssuch as, serum, insulin, transferrin, and epidermal growth factor; (3)protein and tissue hydrolysates, for example peptone or peptone mixtureswhich can be obtained from purified gelatin, plant material, or animalbyproducts; (4) nucleosides and bases such as, adenosine, thymidine, andhypoxanthine; (5) buffers, such as HEPES; (6) antibiotics, such asgentamycin or ampicillin; (7) cell protective agents, for examplepluronic polyol; and (8) galactose. In one embodiment, soluble factorscan be added to the culturing medium.

The mammalian cell culture that can be used with the present inventionis prepared in a medium suitable for the type of cell being cultured. Inone embodiment, the cell culture medium can be any one of thosepreviously discussed (for example, MEM) that is supplemented with serumfrom a mammalian source (for example, fetal bovine serum (FBS)). Inanother embodiment, the medium can be a conditioned medium to sustainthe growth of host cells.

Three-dimensional cultures can be formed from agar (such as Gey's Agar),hydrogels (such as matrigel, agarose, and the like; Lee et al., (2004)Biomaterials 25: 2461-2466) or polymers that are cross-linked. Thesepolymers can comprise natural polymers and their derivatives, syntheticpolymers and their derivatives, or a combination thereof. Naturalpolymers can be anionic polymers, cationic polymers, amphipathicpolymers, or neutral polymers. Non-limiting examples of anionic polymerscan include hyaluronic acid, alginic acid (alginate), carageenan,chondroitin sulfate, dextran sulfate, and pectin. Some examples ofcationic polymers, include but are not limited to, chitosan orpolylysine. (Peppas et al., (2006) Adv Mater. 18: 1345-60; Hoffman, A.S., (2002) Adv Drug Deliv Rev. 43: 3-12; Hoffman, A. S., (2001) Ann NYAcad Sci 944: 62-73). Examples of amphipathic polymers can include, butare not limited to collagen, gelatin, fibrin, and carboxymethyl chitin.Non-limiting examples of neutral polymers can include dextran, agarose,or pullulan. (Peppas et al., (2006) Adv Mater. 18: 1345-60; Hoffman, A.S., (2002) Adv Drug Deliv Rev. 43: 3-12; Hoffman, A. S., (2001) Ann NYAcad Sci 944: 62-73).

Cells to be cultured can harbor introduced expression vectors, such asplasmids. The expression vector constructs can be introduced viatransformation, microinjection, transfection, lipofection,electroporation, or infection. The expression vectors can contain codingsequences, or portions thereof, encoding the proteins for expression andproduction. Expression vectors containing sequences encoding theproduced proteins and polypeptides, as well as the appropriatetranscriptional and translational control elements, can be generatedusing methods well known to and practiced by those skilled in the art.These methods include synthetic techniques, in vitro recombinant DNAtechniques, and in vivo genetic recombination which are described in J.Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Press, Plainview, N.Y. and in F. M. Ausubel et al., 1989,Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y.

FGFR Fusion Molecule Inhibitors

The invention provides methods for use of compounds that decrease theexpression level or activity of a FGFR fusion molecule in a subject. Inaddition, the invention provides methods for using compounds for thetreatment of a gene-fusion associated cancer. In one embodiment, thegene-fusion associated cancer is an epithelial cancer. In oneembodiment, the gene-fusion associated cancer comprises glioblastomamultiforme, breast cancer, lung cancer, prostate cancer, or colorectalcarcinoma.

As used herein, a “FGFR fusion molecule inhibitor” refers to a compoundthat interacts with a FGFR fusion molecule of the invention andmodulates its activity and/or its expression. For example, the compoundcan decrease the activity or expression of a FGFR fusion molecule. Thecompound can be an antagonist of a FGFR fusion molecule (e.g., a FGFRfusion molecule inhibitor). Some non-limiting examples of FGFR fusionmolecule inhibitors include peptides (such as peptide fragmentscomprising a FGFR fusion molecule, or antibodies or fragments thereof),small molecules, and nucleic acids (such as siRNA or antisense RNAspecific for a nucleic acid comprising a FGFR fusion molecule).Antagonists of a FGFR fusion molecule decrease the amount or theduration of the activity of an FGFR fusion protein. In one embodiment,the fusion protein comprises a tyrosine kinase domain of an FGFR proteinfused to a polypeptide that constitutively activates the tyrosine kinasedomain of the FGFR protein (e.g., FGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3or other FGFR-TACC), or a fusion protein comprises a transforming acidiccoiled-coil (TACC) domain fused to a polypeptide with a tyrosine kinasedomain, wherein the TACC domain constitutively activates the tyrosinekinase domain. Antagonists include proteins, nucleic acids, antibodies,small molecules, or any other molecule which decrease the activity of aFGFR fusion molecule.

The term “modulate,” as it appears herein, refers to a change in theactivity or expression of a FGFR fusion molecule. For example,modulation can cause a decrease in protein activity, bindingcharacteristics, or any other biological, functional, or immunologicalproperties of a FGFR fusion molecule, such as an FGFR fusion protein.

In one embodiment, a FGFR fusion molecule inhibitor can be a peptidefragment of a FGFR fusion protein that binds to the protein itself.

For example, the FGFR fusion polypeptide can encompass any portion of atleast about 8 consecutive amino acids of SEQ ID NOS: 79, 88, 150,158-161, or 539-547. The fragment can comprise at least about 10consecutive amino acids, at least about 20 consecutive amino acids, atleast about 30 consecutive amino acids, at least about 40 consecutiveamino acids, a least about 50 consecutive amino acids, at least about 60consecutive amino acids, at least about 70 consecutive amino acids, atleast about 75 consecutive amino acids, at least about 80 consecutiveamino acids, at least about 85 consecutive amino acids, at least about90 consecutive amino acids, at least about 95 consecutive amino acids,at least about 100 consecutive amino acids, at least about 200consecutive amino acids, at least about 300 consecutive amino acids, atleast about 400 consecutive amino acids, at least about 500 consecutiveamino acids, at least about 600 consecutive amino acids, at least about700 consecutive amino acids, or at least about 800 consecutive aminoacids of SEQ ID NOS: 79, 88, 150, 158-161, or 539-547. Fragments includeall possible amino acid lengths between about 8 and 100 about aminoacids, for example, lengths between about 10 and about 100 amino acids,between about 15 and about 100 amino acids, between about 20 and about100 amino acids, between about 35 and about 100 amino acids, betweenabout 40 and about 100 amino acids, between about 50 and about 100 aminoacids, between about 70 and about 100 amino acids, between about 75 andabout 100 amino acids, or between about 80 and about 100 amino acids.These peptide fragments can be obtained commercially or synthesized vialiquid phase or solid phase synthesis methods (Atherton et al., (1989)Solid Phase Peptide Synthesis: a Practical Approach. IRL Press, Oxford,England). The FGFR fusion peptide fragments can be isolated from anatural source, genetically engineered, or chemically prepared. Thesemethods are well known in the art.

A FGFR fusion molecule inhibitor can be a protein, such as an antibody(monoclonal, polyclonal, humanized, chimeric, or fully human), or abinding fragment thereof, directed against a FGFR fusion molecule of theinvention. An antibody fragment can be a form of an antibody other thanthe full-length form and includes portions or components that existwithin full-length antibodies, in addition to antibody fragments thathave been engineered. Antibody fragments can include, but are notlimited to, single chain Fv (scFv), diabodies, Fv, and (Fab′)₂,triabodies, Fc, Fab, CDR1, CDR2, CDR3, combinations of CDR's, variableregions, tetrabodies, bifunctional hybrid antibodies, framework regions,constant regions, and the like (see, Maynard et al., (2000) Ann. Rev.Biomed. Eng. 2:339-76; Hudson (1998) Curr. Opin. Biotechnol. 9:395-402).Antibodies can be obtained commercially, custom generated, orsynthesized against an antigen of interest according to methodsestablished in the art (see U.S. Pat. Nos. 6,914,128, 5,780,597,5,811,523; Roland E. Kontermann and Stefan Dübel (editors), AntibodyEngineering, Vol. I & II, (2010) 2^(nd) ed., Springer; Antony S.Dimitrov (editor), Therapeutic Antibodies: Methods and Protocols(Methods in Molecular Biology), (2009), Humana Press; Benny Lo (editor)Antibody Engineering: Methods and Protocols (Methods in MolecularBiology), (2004) Humana Press, each of which are hereby incorporated byreference in their entireties). For example, antibodies directed to aFGFR fusion molecule can be obtained commercially from Abcam, Santa CruzBiotechnology, Abgent, R&D Systems, Novus Biologicals, etc. Humanantibodies directed to a FGFR fusion molecule (such as monoclonal,humanized, fully human, or chimeric antibodies) can be useful antibodytherapeutics for use in humans. In one embodiment, an antibody orbinding fragment thereof is directed against SEQ ID NOS: 79, 88, 150,158-161, or 539-547.

Inhibition of RNA encoding a FGFR fusion molecule can effectivelymodulate the expression of a FGFR fusion molecule. Inhibitors areselected from the group comprising: siRNA; interfering RNA or RNAi;dsRNA; RNA Polymerase III transcribed DNAs; ribozymes; and antisensenucleic acids, which can be RNA, DNA, or an artificial nucleic acid.

Antisense oligonucleotides, including antisense DNA, RNA, and DNA/RNAmolecules, act to directly block the translation of mRNA by binding totargeted mRNA and preventing protein translation. For example, antisenseoligonucleotides of at least about 15 bases and complementary to uniqueregions of the DNA sequence encoding a FGFR fusion molecule can besynthesized, e.g., by conventional phosphodiester techniques (Dallas etal., (2006) Med. Sci. Monit. 12(4):RA67-74; Kalota et al., (2006) Handb.Exp. Pharmacol. 173:173-96; Lutzelburger et al., (2006) Handb. Exp.Pharmacol. 173:243-59). Antisense nucleotide sequences include, but arenot limited to: morpholinos, 2′-O-methyl polynucleotides, DNA, RNA andthe like.

siRNA comprises a double stranded structure containing from about 15 toabout 50 base pairs, for example from about 21 to about 25 base pairs,and having a nucleotide sequence identical or nearly identical to anexpressed target gene or RNA within the cell. The siRNA comprise a senseRNA strand and a complementary antisense RNA strand annealed together bystandard Watson-Crick base-pairing interactions. The sense strandcomprises a nucleic acid sequence which is substantially identical to anucleic acid sequence contained within the target miRNA molecule.“Substantially identical” to a target sequence contained within thetarget mRNA refers to a nucleic acid sequence that differs from thetarget sequence by about 3% or less. The sense and antisense strands ofthe siRNA can comprise two complementary, single-stranded RNA molecules,or can comprise a single molecule in which two complementary portionsare base-paired and are covalently linked by a single-stranded “hairpin”area. See also, McMnaus and Sharp (2002) Nat Rev Genetics, 3:737-47, andSen and Blau (2006) FASEB J., 20:1293-99, the entire disclosures ofwhich are herein incorporated by reference.

The siRNA can be altered RNA that differs from naturally-occurring RNAby the addition, deletion, substitution and/or alteration of one or morenucleotides. Such alterations can include addition of non-nucleotidematerial, such as to the end(s) of the siRNA or to one or more internalnucleotides of the siRNA, or modifications that make the siRNA resistantto nuclease digestion, or the substitution of one or more nucleotides inthe siRNA with deoxyribo-nucleotides. One or both strands of the siRNAcan also comprise a 3′ overhang. As used herein, a 3′ overhang refers toat least one unpaired nucleotide extending from the 3′-end of a duplexedRNA strand. For example, the siRNA can comprise at least one 3′ overhangof from 1 to about 6 nucleotides (which includes ribonucleotides ordeoxyribonucleotides) in length, or from 1 to about 5 nucleotides inlength, or from 1 to about 4 nucleotides in length, or from about 2 toabout 4 nucleotides in length. For example, each strand of the siRNA cancomprise 3′ overhangs of dithymidylic acid (“TT”) or diuridylic acid(“uu”).

siRNA can be produced chemically or biologically, or can be expressedfrom a recombinant plasmid or viral vector (for example, see U.S. Pat.No. 7,294,504 and U.S. Pat. No. 7,422,896, the entire disclosures ofwhich are herein incorporated by reference). Exemplary methods forproducing and testing dsRNA or siRNA molecules are described in U.S.Patent Application Publication No. 2002/0173478 to Gewirtz, U.S. Pat.No. 8,071,559 to Hannon et al., and in U.S. Pat. No. 7,148,342 toTolentino et al., the entire disclosures of which are hereinincorporated by reference.

In one embodiment, an siRNA directed to a human nucleic acid sequencecomprising a FGFR fusion molecule can be generated against any one ofSEQ ID NOS: 80-82, 84, 94-145, 515, 517, 519-527 or 530-538. In anotherembodiment, an siRNA directed to a human nucleic acid sequencecomprising a breakpoint of an FGFR fusion molecule can be generatedagainst any one of SEQ ID NOS: 1-77, 80-82, 84-145, 515, 517, 519-527 or530-538. In one embodiment, the hairpin sequences targeting the FGFR3gene comprise SEQ ID NOS: 182, 183, or 184.

RNA polymerase III transcribed DNAs contain promoters, such as the U6promoter. These DNAs can be transcribed to produce small hairpin RNAs inthe cell that can function as siRNA or linear RNAs, which can functionas antisense RNA. The FGFR fusion molecule inhibitor can compriseribonucleotides, deoxyribonucleotides, synthetic nucleotides, or anysuitable combination such that the target RNA and/or gene is inhibited.In addition, these forms of nucleic acid can be single, double, triple,or quadruple stranded. (see for example Bass (2001) Nature, 411:428-429;Elbashir et al., (2001) Nature, 411:494 498; U.S. Pat. No. 6,509,154;U.S. Patent Application Publication No. 2003/0027783; and PCTPublication Nos. WO 00/044895, WO 99/032619, WO 00/01846, WO 01/029058,WO 00/044914).

FGFR fusion molecule inhibitor can be a small molecule that binds to aFGFR fusion protein described herein and disrupts its function. Smallmolecules are a diverse group of synthetic and natural substancesgenerally having low molecular weights. They can be isolated fromnatural sources (for example, plants, fungi, microbes and the like), areobtained commercially and/or available as libraries or collections, orsynthesized. Candidate small molecules that inhibit a FGFR fusionprotein can be identified via in silico screening or high-through-put(HTP) screening of combinatorial libraries according to methodsestablished in the art (e.g., see Potyrailo et al., (2011) ACS Comb Sci.13(6):579-633; Mensch et al., (2009) J Pharm Sci. 98(12):4429-68; Schnur(2008) Curr Opin Drug Discov Devel. 11(3):375-80; and Jhoti (2007) ErnstSchering Found Symp Proc. (3):169-85, each of which are herebyincorporated by reference in their entireties.) Most conventionalpharmaceuticals, such as aspirin, penicillin, and manychemotherapeutics, are small molecules, can be obtained commercially,can be chemically synthesized, or can be obtained from random orcombinatorial libraries as described below (see, e.g., Werner et al.,(2006) Brief Funct. Genomic Proteomic 5(1):32-6).

Non-limiting examples of FGFR fusion molecule inhibitors include theFGFR inhibitors AZD4547 (see Gavine et al., (2012) Cancer Res, 72(8);2045-56; see also PCT Application Publication No. WO 2008/075068, eachof which are hereby incorporated by reference in their entireties);NVP-BGJ398 (see Guagnano et al., (2011) J. Med. Chem., 54:7066-7083; seealso U.S. Patent Application Publication No. 2008-0312248 A1, each ofwhich are hereby incorporated by reference in their entireties);PD173074 (see Guagnano et al., (2011) J. Med. Chem., 54:7066-7083; seealso Mohammadi et al., (1998) EMBO J., 17:5896-5904, each of which arehereby incorporated by reference in their entireties); NF449 (EMDMillipore (Billerica, Mass.) Cat. No. 480420; see also Krejci, (2010)the Journal of Biological Chemistry, 285(27):20644-20653, which ishereby incorporated by reference in its entirety); LY2874455 (ActiveBiochem; see Zhao et al. (2011) Mol Cancer Ther. (11):2200-10; see alsoPCT Application Publication No. WO 2010129509, each of which are herebyincorporated by reference in their entireties); TKI258 (Dovitinib);BIBF-1120 (Intedanib-Vargatef); BMS-582664 (Brivanib alaninate);AZD-2171 (Cediranib); TSU-68 (Orantinib); AB-1010 (Masitinib); AP-24534(Ponatinib); and E-7080 (by Eisai). A non-limiting example of an FGFRfusion molecule inhibitor includes the TACC inhibitor KHS101 (Wurdak etal., (2010) PNAS, 107(38): 16542-47, which is hereby incorporated byreference in its entirety).

Structures of FGFR fusion molecule inhibitors useful for the inventioninclude, but are not limited to: the FGFR inhibitor AZD4547,

the FGFR inhibitor NVP-BGJ398,

the FGFR inhibitor PD173074,

the FGFR inhibitor LY2874455

and the FGFR inhibitor NF449 (EMD Millipore (Billerica, Mass.) Cat. No.480420),

Other FGFR inhibitors include, but are not limited to:

In other embodiments, the FGFR fusion molecule inhibitor comprises anoral pan-FGFR tyrosine kinase inhibitor. In other embodiments, the FGFRfusion molecule inhibitor comprises JNJ-42756493. Structures of FGFRfusion molecule inhibitors useful for the invention include, but are notlimited to: the FGFR inhibitor JNJ-42756493.

A structure of an FGFR fusion molecule inhibitor useful for theinvention include, but is not limited to the TACC inhibitor KHS101,

Assessment and Therapeutic Treatment

The invention provides a method of decreasing the growth of a solidtumor in a subject. The tumor is associated with, but not limited to,glioblastoma multiforme, breast cancer, lung cancer, prostate cancer, orcolorectal carcinoma. In another embodiment, the tumor is associatedwith, but not limited to, bladder carcinoma, squamous lung carcinoma andhead and neck carcinoma. In one embodiment, the tumor is associatedwith, but not limited to, glioma. In one embodiment, the tumor isassociated with, but not limited to, grade II or III glioma. In oneembodiment, the tumor is associated with, but not limited to, IDHwild-type grade II or III glioma. In one embodiment, the methodcomprises detecting the presence of a FGFR fusion molecule in a sampleobtained from a subject. In some embodiments, the sample is incubatedwith an agent that binds to an FGFR fusion molecule, such as anantibody, a probe, a nucleic acid primer, and the like. In furtherembodiments, the method comprises administering to the subject aneffective amount of a FGFR fusion molecule inhibitor, wherein theinhibitor decreases the size of the solid tumor. In further embodiments,the method comprises further detecting the presence of IDH1 mutations,EGFR amplification, CDK4 amplification, or MDM2 amplification. Infurther embodiments, a FGFR fusion molecule inhibitor can beadministered in combination with CDK4 inhibitors, MDM2 inhibitors, or acombination thereof.

The invention also provides a method for treating or preventing agene-fusion associated cancer in a subject. In one embodiment, thegene-fusion associated cancer comprises an epithelial cancer. In oneembodiment, the gene-fusion associated cancer comprises glioblastomamultiforme, breast cancer, lung cancer, prostate cancer, or colorectalcarcinoma. In some embodiments, the epithelial cancer comprises bladderurothelial carcinoma, breast carcinoma, colorectal cancer, prostatecarcinoma, lung squamous cell carcinoma, head and neck squamous cellcarcinoma, or a combination of the epithelial cancers described. In oneembodiment, the gene-fusion associated cancer comprises glioma. In oneembodiment, the gene-fusion associated cancer comprises grade II or IIIglioma. In one embodiment, the gene-fusion associated cancer comprisesIDH wild-type grade II or III glioma. In one embodiment, the methodcomprises detecting the presence of a FGFR fusion molecule in a sampleobtained from a subject, the presence of the fusion being indicative ofa gene-fusion associated cancer, and, administering to the subject inneed a therapeutic treatment against a gene-fusion associated cancer. Insome embodiments, the sample is incubated with an agent that binds to anFGFR fusion molecule, such as an antibody, a probe, a nucleic acidprimer, and the like. In further embodiments, the method comprisesfurther detecting the presence of IDH1 mutations, EGFR amplification,CDK4 amplification, or MDM2 amplification. In further embodiments, anagent that binds to an FGFR fusion molecule can be administered incombination with CDK4 inhibitors, MDM2 inhibitors, or a combinationthereof.

The invention also provides a method for decreasing in a subject in needthereof the expression level or activity of a fusion protein comprisingthe tyrosine kinase domain of an FGFR protein fused to a polypeptidethat constitutively activates the tyrosine kinase domain of the FGFRprotein. In some embodiments, the method comprises obtaining abiological sample from the subject. In some embodiments, the sample isincubated with an agent that binds to an FGFR fusion molecule, such asan antibody, a probe, a nucleic acid primer, and the like. In someembodiments, the method comprises administering to the subject atherapeutic amount of a composition comprising an admixture of apharmaceutically acceptable carrier an inhibitor of the fusion proteinof the invention. In one embodiment, the inhibitor is JNJ-42756493. Inanother embodiment, the method further comprises determining the fusionprotein expression level or activity. In another embodiment, the methodfurther comprises detecting whether the fusion protein expression levelor activity is decreased as compared to the fusion protein expressionlevel or activity prior to administration of the composition, therebydecreasing the expression level or activity of the fusion protein. Insome embodiments, the fusion protein is an FGFR-TACC fusion protein. Infurther embodiments, the method comprises further detecting the presenceof IDH1 mutations, EGFR amplification, CDK4 amplification, or MDM2amplification.

The administering step in each of the claimed methods can comprise adrug administration, such as FGFR fusion molecule inhibitor (forexample, a pharmaceutical composition comprising an antibody thatspecifically binds to a FGFR fusion molecule or a fragment thereof; asmall molecule that specifically binds to a FGFR protein; a smallmolecule that specifically binds to a TACC protein; an antisense RNA orantisense DNA that decreases expression of a FGFR fusion molecule; asiRNA that specifically targets a gene encoding a FGFR fusion molecule;a small molecule such as JNJ-42756493; or a combination thereof). In oneembodiment, the therapeutic molecule to be administered comprises apolypeptide of a FGFR fusion molecule, comprising at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about93%, at least about 95%, at least about 97%, at least about 98%, atleast about 99%, or 100% of the amino acid sequence of SEQ ID NOS: 79,88, 150, 158-161, or 539-547 and exhibits the function of decreasingexpression of such a protein, thus treating a gene fusion-associatedcancer. In another embodiment, administration of the therapeuticmolecule decreases the size of the solid tumor associated withglioblastoma multiforme, breast cancer, lung cancer, prostate cancer,colorectal carcinoma, bladder carcinoma, squamous lung carcinoma andhead and neck carcinoma, glioma, grade II or III glioma, or IDHwild-type grade II or III glioma. In further embodiments, thetherapeutic molecule can be administered in combination with CDK4inhibitors, MDM2 inhibitors, or a combination thereof.

In another embodiment, the therapeutic molecule to be administeredcomprises an siRNA directed to a human nucleic acid sequence comprisinga FGFR fusion molecule. In one embodiment, the siRNA is directed to anyone of SEQ ID NOS: 80-82, 84, 94-145, 515, 517, 519-527, or 530-538. Inanother embodiment, the siRNA is directed to any one of SEQ ID NOS:1-77, 80-82, 84-145, 515, 517, 519-527, or 530-538. In a furtherembodiment, the therapeutic molecule to be administered comprises anantibody or binding fragment thereof, which is directed against SEQ IDNOS: 79, 88, 150, 158-161, or 539-547. In some embodiments, thetherapeutic molecule to be administered comprises a small molecule thatspecifically binds to a FGFR protein, such as AZD4547, NVP-BGJ398,PD173074, NF449, TK1258, BIBF-1120, BMS-582664, AZD-2171, TSU68, AB1010,AP24534, E-7080, or LY2874455. In some embodiments, the therapeuticmolecule to be administered is JNJ-42756493. In other embodiments, thetherapeutic molecule to be administered comprises a small molecule thatspecifically binds to a TACC protein, such as KHS101.

In one embodiment, the invention provides for the detection of achromosomal rearrangement at given chromosomal coordinates. In anotherembodiment, the detection or determination comprises nucleic acidsequencing, selective hybridization, selective amplification, geneexpression analysis, or a combination thereof. In another embodiment,the detection or determination comprises protein expression analysis,for example by western blot analysis, immunostaining, ELISA, or otherantibody detection methods.

In one embodiment, the biological sample comprises neuronal cells,serum, bone marrow, blood, peripheral blood, lymph nodes, cerebro-spinalfluid, urine, a saliva sample, a buccal swab, a serum sample, a sputumsample, a lacrimal secretion sample, a semen sample, a vaginal secretionsample, a fetal tissue sample, or a combination thereof. In someembodiments the sample is a tissue sample. In some embodiments, thesample is a paraffin embedded tissue section. In some embodiments, thetissue sample is a tumor sample.

A FGFR fusion molecule, for example, a fusion between FGFR1, FGFR2,FGFR3, or any other FGFR, and TACC1, TACC2, TACC3 or any other TACC, canbe determined at the level of the DNA, RNA, or polypeptide. Optionally,detection can be determined by performing an oligonucleotide ligationassay, a confirmation based assay, a hybridization assay, a sequencingassay, an allele-specific amplification assay, a microsequencing assay,a melting curve analysis, a denaturing high performance liquidchromatography (DHPLC) assay (for example, see Jones et al, (2000) HumGenet., 106(6):663-8), or a combination thereof. In one embodiment, thedetection is performed by sequencing all or part of a FGFR fusionmolecule (e.g., a FGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3 or otherFGFR-TACC nucleic acid, or a FGFR1, TACC1, FGFR2, TACC2, FGFR3, TACC3 orother FGFR or TACC nucleic acid), or by selective hybridization oramplification of all or part of a FGFR fusion molecule (e.g., aFGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3 or other FGFR-TACC nucleic acid,or a FGFR1, TACC1, FGFR2, TACC2, FGFR3, TACC3 or other FGFR or TACCnucleic acid). A FGFR fusion molecule specific amplification (e.g., aFGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3 or other FGFR-TACC nucleic acidspecific amplification) can be carried out before the fusionidentification step.

The invention provides for a method of detecting a chromosomalalteration in a subject afflicted with a gene-fusion associated cancer.In one embodiment, the chromosomal alteration is an in-frame fusedtranscript described herein, for example an FGFR fusion molecule. Insome embodiments, the chromosomal alteration is a chromosomaltranslocation, for example an FGFR fusion molecule. An alteration in achromosome region occupied by a FGFR fusion molecule, such as aFGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3 or other FGFR-TACC nucleic acid,can be any form of mutation(s), deletion(s), rearrangement(s) and/orinsertions in the coding and/or non-coding region of the locus, alone orin various combination(s). Mutations can include point mutations.Insertions can encompass the addition of one or several residues in acoding or non-coding portion of the gene locus. Insertions can comprisean addition of between 1 and 50 base pairs in the gene locus. Deletionscan encompass any region of one, two or more residues in a coding ornon-coding portion of the gene locus, such as from two residues up tothe entire gene or locus. Deletions can affect smaller regions, such asdomains (introns) or repeated sequences or fragments of less than about50 consecutive base pairs, although larger deletions can occur as well.Rearrangement includes inversion of sequences. The alteration in achromosome region occupied by a FGFR fusion molecule, e.g., aFGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3 or other FGFR-TACC nucleic acid,can result in amino acid substitutions, RNA splicing or processing,product instability, the creation of stop codons, production ofoncogenic fusion proteins, frame-shift mutations, and/or truncatedpolypeptide production. The alteration can result in the production of aFGFR fusion molecule, for example, one encoded by a FGFR1-TACC1,FGFR2-TACC2, FGFR3-TACC3 or other FGFR-TACC nucleic acid, with alteredfunction, stability, targeting or structure. The alteration can alsocause a reduction, or even an increase in protein expression. In oneembodiment, the alteration in the chromosome region occupied by a FGFRfusion molecule can comprise a chromosomal rearrangement resulting inthe production of a FGFR fusion molecule, such as a FGFR1-TACC1,FGFR2-TACC2, FGFR3-TACC3 or other FGFR-TACC fusion. This alteration canbe determined at the level of the DNA, RNA, or polypeptide. In anotherembodiment, the detection or determination comprises nucleic acidsequencing, selective hybridization, selective amplification, geneexpression analysis, or a combination thereof. In another embodiment,the detection or determination comprises protein expression analysis,for example by western blot analysis, ELISA, immunostaining or otherantibody detection methods. In one embodiment, the coordinatescomprising FGFR1 translocations comprise chr8:38,268,656-38,325,363. Inanother embodiment, the coordinates comprising FGFR2 translocationscomprise chr10:123,237,844-123,357,972. In a further embodiment, thecoordinates comprising FGFR3 translocations comprisechr4:1,795,039-1,810,599. In yet another embodiment, the coordinatescomprising FGFR4 translocations comprise chr5:176,513,921-176,525,126.In one embodiment, the coordinates comprising TACC1 translocationscomprise chr8:38,644,722-38,710,546. In another embodiment, thecoordinates comprising TACC2 translocations comprisechr10:123,748,689-124,014,057. In a further embodiment, the coordinatescomprising TACC3 translocations comprise chr4:1,723,217-1,746,905.

The present invention provides a method for treating a gene-fusionassociated cancer in a subject in need thereof. In one embodiment, themethod comprises obtaining a sample from the subject to determine thelevel of expression of an FGFR fusion molecule in the subject. In someembodiments, the sample is incubated with an agent that binds to an FGFRfusion molecule, such as an antibody, a probe, a nucleic acid primer,and the like. In another embodiment, the detection or determinationcomprises nucleic acid sequencing, selective hybridization, selectiveamplification, gene expression analysis, or a combination thereof. Inanother embodiment, the detection or determination comprises proteinexpression analysis, for example by western blot analysis,immunostaining, ELISA, or other antibody detection methods. In someembodiments, the method further comprises assessing whether toadminister a FGFR fusion molecule inhibitor based on the expressionpattern of the subject. In further embodiments, the method comprisesadministering a FGFR fusion molecule inhibitor to the subject. In oneembodiment, the FGFR fusion molecule inhibitor is JNJ-42756493. In oneembodiment, the gene-fusion associated cancer comprises an epithelialcancer. In one embodiment, the gene-fusion associated cancer comprisesglioblastoma multiforme, breast cancer, lung cancer, prostate cancer, orcolorectal carcinoma. In some embodiments, the epithelial cancercomprises bladder urothelial carcinoma, breast carcinoma, colorectalcancer, prostate carcinoma, lung squamous cell carcinoma, head and necksquamous cell carcinoma, or a combination of the epithelial cancersdescribed. In one embodiment, the gene-fusion associated cancercomprises glioma, grade II or III glioma, or IDH wild-type grade II orIII glioma. In further embodiments, the method comprises furtherdetecting the presence of IDH1 mutations, EGFR amplification, CDK4amplification, or MDM2 amplification. In further embodiments, a FGFRfusion molecule inhibitor can be administered in combination with CDK4inhibitors, MDM2 inhibitors, or a combination thereof.

In one embodiment, the invention provides for a method of detecting thepresence of altered RNA expression of an FGFR fusion molecule in asubject, for example one afflicted with a gene-fusion associated cancer.In another embodiment, the invention provides for a method of detectingthe presence of an FGFR fusion molecule in a subject. In someembodiments, the method comprises obtaining a sample from the subject todetermine whether the subject expresses an FGFR fusion molecule. In someembodiments, the sample is incubated with an agent that binds to an FGFRfusion molecule, such as an antibody, a probe, a nucleic acid primer,and the like. In other embodiments, the detection or determinationcomprises nucleic acid sequencing, selective hybridization, selectiveamplification, gene expression analysis, or a combination thereof. Inanother embodiment, the detection or determination comprises proteinexpression analysis, for example by western blot analysis, ELISA, orother antibody detection methods. In some embodiments, the methodfurther comprises assessing whether to administer a FGFR fusion moleculeinhibitor based on the expression pattern of the subject. In furtherembodiments, the method comprises administering a FGFR fusion moleculeinhibitor to the subject. In one embodiment, the FGFR fusion moleculeinhibitor is JNJ-42756493. Altered RNA expression includes the presenceof an altered RNA sequence, the presence of an altered RNA splicing orprocessing, or the presence of an altered quantity of RNA. These can bedetected by various techniques known in the art, including sequencingall or part of the RNA or by selective hybridization or selectiveamplification of all or part of the RNA. In a further embodiment, themethod can comprise detecting the presence or expression of a FGFRfusion molecule, such as one encoded by a FGFR1-TACC1, FGFR2-TACC2,FGFR3-TACC3 or other FGFR-TACC nucleic acid. Altered polypeptideexpression includes the presence of an altered polypeptide sequence, thepresence of an altered quantity of polypeptide, or the presence of analtered tissue distribution. These can be detected by various techniquesknown in the art, including by sequencing and/or binding to specificligands (such as antibodies). In one embodiment, the detecting comprisesusing a northern blot; real time PCR and primers directed to SEQ ID NOS:80-82, 84, 94-145, 515, 517, 519-527, or 530-538; a ribonucleaseprotection assay; a hybridization, amplification, or sequencingtechnique to detect an FGFR fusion molecule, such as one comprising SEQID NOS: 80-82, 84, 94-145, 515, 517, 519-527, or 530-538; or acombination thereof. In another embodiment, the PCR primers comprise SEQID NOS: 162, 163, 164, 165 166, 167, 168, 169, 495, 496, 497, 498, 507,508, 509, 510, 511, 512, 513, or 514. In a further embodiment, primersused for the screening of FGFR fusion molecules, such as FGFR-TACCfusions, comprise SEQ ID NOS: 166, 167, 168, 169, 495, 496, 497, 498,507, 508, 509, or 510. In some embodiments, primers used for genomicdetection of an FGFR3-TACC3 fusion comprise SEQ ID NOS: 170 171, 499,500, 501, 502, 503, 504, 505, or 506.

In some aspects of the invention, the method comprises further detectingthe presence of IDH1 mutations, EGFR amplification, CDK4 amplification,or MDM2 amplification. MDM2 encodes a nuclear-localized E3 ubiquitinligase. Alternative splicing results in a multitude of transcriptvariants, many of which may be expressed only in tumor cells. EGFR(epidermal growth factor receptor) is a transmembrane glycoprotein thatis a member of the protein kinase superfamily. This protein is areceptor for members of the epidermal growth factor family. EGFR is acell surface protein that binds to epidermal growth factor. Multiplealternatively spliced transcript variants that encode different proteinisoforms have been found for this gene. CDK4 (cyclin dependent kinase 4)is a member of the Ser/Thr protein kinase family. It is a catalyticsubunit of the protein kinase complex that is important for cell cycleG1 phase progression. The activity of this kinase is restricted to theG1-S phase, which is controlled by the regulatory subunits D-typecyclins and CDK inhibitor p16(INK4a). Multiple polyadenylation sites ofthis gene have been reported. IDH1 (isocitrate dehydrogenase 1 (NADP+),soluble) catalyzes the oxidative decarboxylation of isocitrate to2-oxoglutarate. Alternatively spliced transcript variants encoding thesame protein have been found for this gene. IDH1 mutations, EGFRamplification, CDK4 amplification, or MDM2 amplification can be detectedusing various techniques know in the art, including, but not limited tosequencing and qPCR.

Various techniques known in the art can be used to detect or quantifyaltered gene or RNA expression or nucleic acid sequences, which include,but are not limited to, hybridization, sequencing, amplification, and/orbinding to specific ligands (such as antibodies). Other suitable methodsinclude allele-specific oligonucleotide (ASO), oligonucleotide ligation,allele-specific amplification, Southern blot (for DNAs), Northern blot(for RNAs), single-stranded conformation analysis (SSCA), PFGE,fluorescent in situ hybridization (FISH), gel migration, clampeddenaturing gel electrophoresis, denaturing HLPC, melting curve analysis,heteroduplex analysis, RNase protection, chemical or enzymatic mismatchcleavage, ELISA, radio-immunoassays (RIA) and immuno-enzymatic assays(IEMA).

Some of these approaches (such as SSCA and constant gradient gelelectrophoresis (CGGE)) are based on a change in electrophoreticmobility of the nucleic acids, as a result of the presence of an alteredsequence. According to these techniques, the altered sequence isvisualized by a shift in mobility on gels. The fragments can then besequenced to confirm the alteration. Some other approaches are based onspecific hybridization between nucleic acids from the subject and aprobe specific for wild type or altered gene or RNA. The probe can be insuspension or immobilized on a substrate. The probe can be labeled tofacilitate detection of hybrids. Some of these approaches are suited forassessing a polypeptide sequence or expression level, such as Northernblot, ELISA and RIA. These latter require the use of a ligand specificfor the polypeptide, for example, the use of a specific antibody.

Hybridization.

Hybridization detection methods are based on the formation of specifichybrids between complementary nucleic acid sequences that serve todetect nucleic acid sequence alteration(s). A detection techniqueinvolves the use of a nucleic acid probe specific for a wild type oraltered gene or RNA, followed by the detection of the presence of ahybrid. The probe can be in suspension or immobilized on a substrate orsupport (for example, as in nucleic acid array or chips technologies).The probe can be labeled to facilitate detection of hybrids. In oneembodiment, the probe according to the invention can comprise a nucleicacid directed to SEQ ID NOS: 80-82, 84, 94-145, 515, 517, 519-527, or530-538. For example, a sample from the subject can be contacted with anucleic acid probe specific for a gene encoding a FGFR fusion molecule,and the formation of a hybrid can be subsequently assessed. In oneembodiment, the method comprises contacting simultaneously the samplewith a set of probes that are specific for an FGFR fusion molecule.Also, various samples from various subjects can be investigated inparallel.

According to the invention, a probe can be a polynucleotide sequencewhich is complementary to and specifically hybridizes with a, or atarget portion of a, gene or RNA corresponding to a FGFR fusionmolecule. Useful probes are those that are complementary to the gene,RNA, or target portion thereof. Probes can comprise single-strandednucleic acids of between 8 to 1000 nucleotides in length, for instancebetween 10 and 800, between 15 and 700, or between 20 and 500. Longerprobes can be used as well. A useful probe of the invention is a singlestranded nucleic acid molecule of between 8 to 500 nucleotides inlength, which can specifically hybridize to a region of a gene or RNAthat corresponds to a FGFR fusion molecule.

The sequence of the probes can be derived from the sequences of the FGFRfusion genes provided herein. Nucleotide substitutions can be performed,as well as chemical modifications of the probe. Such chemicalmodifications can be accomplished to increase the stability of hybrids(e.g., intercalating groups) or to label the probe. Some examples oflabels include, without limitation, radioactivity, fluorescence,luminescence, and enzymatic labeling.

A guide to the hybridization of nucleic acids is found in e.g.,Sambrook, ed., Molecular Cloning: A Laboratory Manual (3^(rd) Ed.),Vols. 1-3, Cold Spring Harbor Laboratory, 1989; Current Protocols InMolecular Biology, Ausubel, ed. John Wiley & Sons, Inc., New York, 2001;Laboratory Techniques In Biochemistry And Molecular Biology:Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic AcidPreparation, Tijssen, ed. Elsevier, N.Y., 1993.

Sequencing.

Sequencing can be carried out using techniques well known in the art,using automatic sequencers. The sequencing can be performed on thecomplete FGFR fusion molecule or on specific domains thereof.

Amplification.

Amplification is based on the formation of specific hybrids betweencomplementary nucleic acid sequences that serve to initiate nucleic acidreproduction. Amplification can be performed according to varioustechniques known in the art, such as by polymerase chain reaction (PCR),ligase chain reaction (LCR), strand displacement amplification (SDA) andnucleic acid sequence based amplification (NASBA). These techniques canbe performed using commercially available reagents and protocols. Usefultechniques in the art encompass real-time PCR, allele-specific PCR, orPCR based single-strand conformational polymorphism (SSCP).Amplification usually requires the use of specific nucleic acid primers,to initiate the reaction. For example, nucleic acid primers useful foramplifying sequences corresponding to a FGFR fusion molecule are able tospecifically hybridize with a portion of the gene locus that flanks atarget region of the locus. In one embodiment, amplification comprisesusing forward and reverse PCR primers directed to SEQ ID NOS: 80-82, 84,94-145, 515, 517, 519-527, or 530-538. Nucleic acid primers useful foramplifying sequences from a FGFR fusion molecule (e.g., a FGFR1-TACC1,FGFR2-TACC2, FGFR3-TACC3 or other FGFR-TACC nucleic acid); the primersspecifically hybridize with a portion of an FGFR fusion molecule. Incertain subjects, the presence of an FGFR fusion molecule corresponds toa subject with a gene fusion-associated cancer. In one embodiment,amplification can comprise using forward and reverse PCR primerscomprising nucleotide sequences of SEQ ID NOS: 80-82, 84, 94-145, 515,517, 519-527, or 530-538. In one embodiment, amplification can compriseusing forward and reverse PCR primers comprising nucleotide sequences ofSEQ ID NOS: 162-169, or 495-514.

Non-limiting amplification methods include, e.g., polymerase chainreaction, PCR (PCR Protocols, A Guide To Methods And Applications, ed.Innis, Academic Press, N.Y., 1990 and PCR Strategies, 1995, ed. Innis,Academic Press, Inc., N.Y.); ligase chain reaction (LCR) (Wu (1989)Genomics 4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene89:117); transcription amplification (Kwoh (1989) PNAS 86:1173); and,self-sustained sequence replication (Guatelli (1990) PNAS 87:1874); QBeta replicase amplification (Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (Burg(1996) Mol. Cell. Probes 10:257-271) and other RNA polymerase mediatedtechniques (e.g., NASBA, Cangene, Mississauga, Ontario; see also Berger(1987) Methods Enzymol. 152:307-316; U.S. Pat. Nos. 4,683,195 and4,683,202; and Sooknanan (1995) Biotechnology 13:563-564). All thereferences stated above are incorporated by reference in theirentireties.

The invention provides for a nucleic acid primer, wherein the primer canbe complementary to and hybridize specifically to a portion of a FGFRfusion molecule, such as a FGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3 orother FGFR-TACC nucleic acid (e.g., DNA or RNA) in certain subjectshaving a gene fusion-associated cancer. In one embodiment, thegene-fusion associated cancer comprises glioblastoma multiforme, breastcancer, lung cancer, prostate cancer, or colorectal carcinoma. Primersof the invention can be specific for fusion sequences in a FGFR1-TACC1,FGFR2-TACC2, FGFR3-TACC3 or other FGFR-TACC nucleic acid (DNA or RNA).By using such primers, the detection of an amplification productindicates the presence of a fusion of a FGFR1 and TACC1, FGFR2 andTACC2, FGFR3 and TACC3 or other FGFR and TACC nucleic acid. Examples ofprimers of this invention can be single-stranded nucleic acid moleculesof about 5 to 60 nucleotides in length, or about 8 to about 25nucleotides in length. The sequence can be derived directly from thesequence of a FGFR fusion molecule, e.g. FGFR1-TACC1, FGFR2-TACC2,FGFR3-TACC3 or other FGFR-TACC nucleic acid. Perfect complementarity isuseful to ensure high specificity; however, certain mismatch can betolerated. For example, a nucleic acid primer or a pair of nucleic acidprimers as described above can be used in a method for detecting thepresence of a gene fusion-associated cancer in a subject. In oneembodiment, primers can be used to detect an FGFR fusion molecule, suchas a primer comprising SEQ ID NOS: 80-82, 84, 94-145, 515, 517, 519-527,or 530-538; or a combination thereof. In another embodiment, the PCRprimers comprise SEQ ID NOS: 162, 163, 164, 165, 166, 167, 168, 169,495, 496, 497, 498, 507, 508, 509, 510, 511, 512, 513, or 514. Inafurther embodiment, primers used for the screening of FGFR fusionmolecules, such as FGFR-TACC fusions, comprise SEQ ID NOS: 166, 167,168, 169, 495, 496, 497, 498, 507, 508, 509, or 510. In someembodiments, primers used for genomic detection of an FGFR3-TACC3 fusioncomprise SEQ ID NOS: 170, 171, 499, 500, 501, 502, 503, 504, 505, or506. In one embodiment, the method can comprise contacting a sample fromthe subject with primers specific for a FGFR fusion molecule, anddetermining the presence of an PCR product. In another embodiment, themethod can comprise contacting a sample from the subject with primerspecific for a FGFR molecule, or a TACC molecule, and determining thepresence of a PCR product. In another embodiment, the primers canrecognize the nucleic acids encoding a FGFR3 C-terminal region, ornucleic acids encoding a TACC3 N-terminal region, or a combinationthereof. In another embodiment, the method can comprise contacting asample from the subject with primers specific for a FGFR molecule, or aTACC molecule, or a FGFR fusion molecule, and determining the amount ofPCR product formed compared to the amount of PCR product formed innon-tumor cells or tissue, wherein an increased amount of PCR productindicates the presence of an FGFR fusion. In one embodiment, primersand/or the PCR product are labeled to enable detection of the PCRproduct. For example, nucleic acid primers useful for amplifyingsequences corresponding to a FGFR fusion molecules can be labeled withfluorescent molecules, radioactive molecules, chemiluminescentmolecules, or affinity molecules (e.g. biotin) which can then bedetected by methods known in the art (e.g. fluorescently labeledstreptavidin). PCR products can also be detected by using dyes that canbe incorporated into newly formed PCR products, such as, but not limitedto, SYBR Green.

Specific Ligand Binding.

As discussed herein, a nucleic acid encoding a FGFR fusion molecule orexpression of a FGFR fusion molecule, can also be detected by screeningfor alteration(s) in a sequence or expression level of a polypeptideencoded by the same. Different types of ligands can be used, such asspecific antibodies. In one embodiment, the sample is contacted with anantibody specific for a polypeptide encoded by a FGFR fusion moleculeand the formation of an immune complex is subsequently determinedVarious methods for detecting an immune complex can be used, such asELISA, immunostaining, radioimmunoassays (RIA) and immuno-enzymaticassays (IEMA).

For example, an antibody can be a polyclonal antibody, a monoclonalantibody, as well as fragments or derivatives thereof havingsubstantially the same antigen specificity. Fragments include Fab,Fab′2, or CDR regions. Derivatives include single-chain antibodies,humanized antibodies, or poly-functional antibodies. An antibodyspecific for a polypeptide encoded by a FGFR fusion molecule can be anantibody that selectively binds such a polypeptide. In one embodiment,the antibody is raised against a polypeptide encoded by a FGFR fusionmolecule (such as FGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3 or otherFGFR-TACC fusion) or an epitope-containing fragment thereof. Althoughnon-specific binding towards other antigens can occur, binding to thetarget polypeptide occurs with a higher affinity and can be reliablydiscriminated from non-specific binding. In one embodiment, the methodcan comprise contacting a sample from the subject with an antibodyspecific for a FGFR fusion molecule, and determining the presence of animmune complex. Optionally, the sample can be contacted to a supportcoated with antibody specific for a FGFR fusion molecule. In oneembodiment, the sample can be contacted simultaneously, or in parallel,or sequentially, with various antibodies specific for different forms ofa FGFR fusion molecule, e.g., FGFR1-TACC1, FGFR2-TACC2, FGFR3-TACC3 orother FGFR-TACC fusion.

In one embodiment, the method can comprise contacting a sample from thesubject with an antibody specific for a FGFR fusion molecule, anddetermining the presence of an immune complex. In another embodiment,the method can comprise contacting a sample from the subject with anantibody specific for a FGFR molecule, or a TACC molecule, anddetermining the presence of an immune complex. In another embodiment,the antibody can recognize the FGFR3 C-terminal region, or the TACC3N-terminal region, or a combination thereof. In another embodiment, theantibody can recognize the FGFR3 C-terminal region, or the TACC3N-terminal region, or a combination thereof. In another embodiment, themethod can comprise contacting a sample from the subject with anantibody specific for a FGFR molecule, or a TACC molecule, or a FGFRfusion molecule, and determining the amount of an immune complex formedcompared to the amount of immune complex formed in non-tumor cells ortissue, wherein an increased amount of an immune complex indicates thepresence of an FGFR fusion.

Detection the formation of a complex between an antibody and a proteincan be performed by a variety of method known in the art. For example,an antibody-protein complex can be detected by using antibodies orsecondary antibodies labeled with fluorescent molecules, chromogenicmolecules, chemiluminescent molecules, radioactive isotopes, or affinitymolecules (e.g. biotin) which can then be detected by methods known inthe art (e.g. fluorescently labeled streptavidin).

The invention also provides for a diagnostic kit comprising products andreagents for detecting in a sample from a subject the presence of a FGFRfusion molecule. The kit can be useful for determining whether a samplefrom a subject exhibits increased or reduced expression of a FGFR fusionmolecule. For example, the diagnostic kit according to the presentinvention comprises any primer, any pair of primers, any nucleic acidprobe and/or any ligand, or any antibody directed specifically to a FGFRfusion molecule. The diagnostic kit according to the present inventioncan further comprise reagents and/or protocols for performing ahybridization, amplification, or antigen-antibody immune reaction. Inone embodiment, the kit can comprise nucleic acid primers thatspecifically hybridize to and can prime a polymerase reaction from aFGFR fusion molecule comprising SEQ ID NOS: 80-82, 84, 94-145, 515, 517,519-527, or 530-538, or a combination thereof. In one embodiment,primers can be used to detect a FGFR fusion molecule, such as a primerdirected to SEQ ID NOS: 80-82, 84, 94-145, 515, 517, 519-527, or530-538; or a combination thereof. In another embodiment, the PCR primercomprises SEQ ID NOS: 162, 163, 164, 165, 166, 167, 168, 169, 495, 496,497, 498, 507, 508, 509, 510, 511, 512, 513, or 514. Ina furtherembodiment, primers used for the screening of FGFR fusion molecules,such as FGFR-TACC fusions, comprise SEQ ID NOS: 166, 167, 168, 169, 495,496, 497, 498, 507, 508, 509, or 510. In some embodiments, primers usedfor genomic detection of an FGFR3-TACC3 fusion comprise SEQ ID NOS: 170,171, 499, 500, 501, 502, 503, 504, 505, or 506. In some embodiments, thekit comprises an antibody that specifically binds to a FGFR fusionmolecule comprising SEQ ID NOS: 79, 85-89, 150, 158-161, or 539-547,wherein the antibody will recognize the protein only when a FGFR fusionmolecule is present. The diagnosis methods can be performed in vitro, exvivo, or in vivo. These methods utilize a sample from the subject inorder to assess the status of a FGFR fusion molecule. The sample can beany biological sample derived from a subject, which contains nucleicacids or polypeptides. Examples of such samples include, but are notlimited to, fluids, tissues, cell samples, organs, and tissue biopsies.Non-limiting examples of samples include blood, liver, plasma, serum,saliva, urine, or seminal fluid. In some embodiments the sample is atissue sample. In some embodiments, the sample is a paraffin embeddedtissue section. In some embodiments, the tissue sample is a tumorsample. The sample can be collected according to conventional techniquesand used directly for diagnosis or stored. The sample can be treatedprior to performing the method, in order to render or improveavailability of nucleic acids or polypeptides for testing. Treatmentsinclude, for instance, lysis (e.g., mechanical, physical, or chemical),centrifugation. The nucleic acids and/or polypeptides can bepre-purified or enriched by conventional techniques, and/or reduced incomplexity. Nucleic acids and polypeptides can also be treated withenzymes or other chemical or physical treatments to produce fragmentsthereof. In one embodiment, the sample is contacted with reagents, suchas probes, primers, or ligands, in order to assess the presence of aFGFR fusion molecule. Contacting can be performed in any suitabledevice, such as a plate, tube, well, or glass. In some embodiments, thecontacting is performed on a substrate coated with the reagent, such asa nucleic acid array or a specific ligand array. The substrate can be asolid or semi-solid substrate such as any support comprising glass,plastic, nylon, paper, metal, or polymers. The substrate can be ofvarious forms and sizes, such as a slide, a membrane, a bead, a column,or a gel. The contacting can be made under any condition suitable for acomplex to be formed between the reagent and the nucleic acids orpolypeptides of the sample.

Nucleic Acid Delivery Methods

Delivery of nucleic acids into viable cells can be effected ex vivo, insitu, or in vivo by use of vectors, such as viral vectors (e.g.,lentivirus, adenovirus, adeno-associated virus, or a retrovirus), or exvivo by use of physical DNA transfer methods (e.g., liposomes orchemical treatments). Non-limiting techniques suitable for the transferof nucleic acid into mammalian cells in vitro include the use ofliposomes, electroporation, microinjection, cell fusion, DEAE-dextran,and the calcium phosphate precipitation method (See, for example,Anderson, Nature, 1998) supplement to 392(6679):25( ). Introduction of anucleic acid or a gene encoding a polypeptide of the invention can alsobe accomplished with extrachromosomal substrates (transient expression)or artificial chromosomes (stable expression). Cells can also becultured ex vivo in the presence of therapeutic compositions of thepresent invention in order to proliferate or to produce a desired effecton or activity in such cells. Treated cells can then be introduced invivo for therapeutic purposes.

Nucleic acids can be inserted into vectors and used as gene therapyvectors. A number of viruses have been used as gene transfer vectors,including papovaviruses, e.g., SV40 (Madzak et al., (1992) J Gen Virol.73(Pt 6):1533-6), adenovirus (Berkner (1992) Curr Top Microbiol Immunol.158:39-66; Berkner (1988) Biotechniques, 6(7):616-29; Gorziglia andKapikian (1992) J Virol. 66(7):4407-12; Quantin et al., (1992) Proc NatlAcad Sci USA. 89(7):2581-4; Rosenfeld et al., (1992) Cell. 68(1):143-55;Wilkinson et al., (1992) Nucleic Acids Res. 20(9):2233-9;Stratford-Perricaudet et al., (1990) Hum Gene Ther. 1(3):241-56),vaccinia virus (Moss (1992) Curr Opin Biotechnol. 3(5):518-22),adeno-associated virus (Muzyczka, (1992) Curr Top Microbiol Immunol.158:97-129; Ohi et al., (1990) Gene. 89(2):279-82), herpesvirusesincluding HSV and EBV (Margolskee (1992) Curr Top Microbiol Immunol.158:67-95; Johnson et al., (1992) Brain Res Mol Brain Res.12(1-3):95-102; Fink et al., (1992) Hum Gene Ther. 3(1):11-9;Breakefield and Geller (1987) Mol Neurobiol. 1(4):339-71; Freese et al.,(1990) Biochem Pharmacol. 40(10):2189-99), and retroviruses of avian(Bandyopadhyay and Temin (1984) Mol Cell Biol. 4(4):749-54; Petropouloset al., (1992) J Virol. 66(6):3391-7), murine (Miller et al. (1992) MolCell Biol. 12(7):3262-72; Miller et al., (1985) J Virol. 55(3):521-6;Sorge et al., (1984) Mol Cell Biol. 4(9):1730-7; Mann and Baltimore(1985) J Virol. 54(2):401-7; Miller et al., (1988) J Virol.62(11):4337-45), and human origin (Shimada et al., (1991) J Clin Invest.88(3):1043-7; Helseth et al., (1990) J Virol. 64(12):6314-8; Page etal., (1990) J Virol. 64(11):5270-6; Buchschacher and Panganiban (1992) JVirol. 66(5):2731-9).

Non-limiting examples of in vivo gene transfer techniques includetransfection with viral (e.g., retroviral) vectors (see U.S. Pat. No.5,252,479, which is incorporated by reference in its entirety) and viralcoat protein-liposome mediated transfection (Dzau et al., (1993) Trendsin Biotechnology 11:205-210), incorporated entirely by reference). Forexample, naked DNA vaccines are generally known in the art; see Brower,(1998) Nature Biotechnology, 16:1304-1305, which is incorporated byreference in its entirety. Gene therapy vectors can be delivered to asubject by, for example, intravenous injection, local administration(see, e.g., U.S. Pat. No. 5,328,470) or by stereotactic injection (see,e.g., Chen, et al., (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). Thepharmaceutical preparation of the gene therapy vector can include thegene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

For reviews of nucleic acid delivery protocols and methods see Andersonet al. (1992) Science 256:808-813; U.S. Pat. Nos. 5,252,479, 5,747,469,6,017,524, 6,143,290, 6,410,010 6,511,847; and U.S. ApplicationPublication No. 2002/0077313, which are all hereby incorporated byreference in their entireties. For additional reviews, see Friedmann(1989) Science, 244:1275-1281; Verma, Scientific American: 68-84 (1990);Miller (1992) Nature, 357: 455-460; Kikuchi et al. (2008) J DermatolSci. 50(2):87-98; Isaka et al. (2007) Expert Opin Drug Deliv.4(5):561-71; Jager et al. (2007) Curr Gene Ther. 7(4):272-83; Waehler etal. (2007) Nat Rev Genet. 8(8):573-87; Jensen et al. (2007) Ann Med.39(2):108-15; Herweijer et al. (2007) Gene Ther. 14(2):99-107; Eliyahuet al. (2005) Molecules 10(1):34-64; and Altaras et al. (2005) AdvBiochem Eng Biotechnol. 99:193-260, all of which are hereby incorporatedby reference in their entireties.

A FGFR fusion nucleic acid can also be delivered in a controlled releasesystem. For example, the FGFR fusion molecule can be administered usingintravenous infusion, an implantable osmotic pump, a transdermal patch,liposomes, or other modes of administration. In one embodiment, a pumpcan be used (see Sefton (1987) Biomed. Eng. 14:201; Buchwald et al.(1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J Med. 321:574). Inanother embodiment, polymeric materials can be used (see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, New York (1984);Ranger and Peppas, (1983) J. Macromol. Sci. Rev. Macromol. Chem. 23:61;see also Levy et al. (1985) Science 228:190; During et al. (1989) Ann.Neurol. 25:351; Howard et al. (1989) J. Neurosurg. 71:105). In yetanother embodiment, a controlled release system can be placed inproximity of the therapeutic target thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlledrelease systems are discussed in the review by Langer (Science (1990)249:1527-1533).

Pharmaceutical Compositions and Administration for Therapy

An inhibitor of the invention can be incorporated into pharmaceuticalcompositions suitable for administration, for example the inhibitor anda pharmaceutically acceptable carrier

A FGFR fusion molecule or inhibitor of the invention (e.g. JNJ-42756493)can be administered to the subject once (e.g., as a single injection ordeposition). Alternatively, a FGFR fusion molecule or inhibitor can beadministered once or twice daily to a subject in need thereof for aperiod of from about two to about twenty-eight days, or from about sevento about ten days. A FGFR fusion molecule or inhibitor can also beadministered once or twice daily to a subject for a period of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof. AFGFR fusion molecule or inhibitor can also be administered in seven toten day repeating cycles (i.e. administration of a FGFR fusion moleculeor inhibitor for seven to ten days, followed by no administration of aFGFR fusion molecule or inhibitor for seven to ten days). Furthermore, aFGFR fusion molecule or inhibitor of the invention can beco-administrated with another therapeutic. Where a dosage regimencomprises multiple administrations, the effective amount of the FGFRfusion molecule or inhibitor administered to the subject can comprisethe total amount of gene product administered over the entire dosageregimen.

A FGFR fusion molecule or inhibitor can be administered to a subject byany means suitable for delivering the FGFR fusion molecule or inhibitorto cells of the subject, such as cancer cells, e.g., glioblastomamultiforme, breast cancer, lung cancer, prostate cancer, colorectalcarcinoma, bladder carcinoma, squamous lung carcinoma, head and neckcarcinoma, glioma, grade II or III glioma, or IDH wild-type grade II orIII glioma. For example, a FGFR fusion molecule or inhibitor can beadministered by methods suitable to transfect cells. Transfectionmethods for eukaryotic cells are well known in the art, and includedirect injection of the nucleic acid into the nucleus or pronucleus of acell; electroporation; liposome transfer or transfer mediated bylipophilic materials; receptor mediated nucleic acid delivery,bioballistic or particle acceleration; calcium phosphate precipitation,and transfection mediated by viral vectors.

The compositions of this invention can be formulated and administered toreduce the symptoms associated with a gene fusion-associated cancer,e.g., glioblastoma multiforme, breast cancer, lung cancer, prostatecancer, colorectal carcinoma, bladder carcinoma, squamous lungcarcinoma, head and neck carcinoma, glioma, grade II or III glioma, orIDH wild-type grade II or III glioma, by any means that produces contactof the active ingredient with the agent's site of action in the body ofa subject, such as a human or animal (e.g., a dog, cat, or horse). Theycan be administered by any conventional means available for use inconjunction with pharmaceuticals, either as individual therapeuticactive ingredients or in a combination of therapeutic activeingredients. They can be administered alone, but are generallyadministered with a pharmaceutical carrier selected on the basis of thechosen route of administration and standard pharmaceutical practice.

A therapeutically effective dose of FGFR fusion molecule or inhibitor(e.g. JNJ-42756493) can depend upon a number of factors known to thoseor ordinary skill in the art. The dose(s) of the FGFR fusion moleculeinhibitor can vary, for example, depending upon the identity, size, andcondition of the subject or sample being treated, further depending uponthe route by which the composition is to be administered, if applicable,and the effect which the practitioner desires the a FGFR fusion moleculeinhibitor to have upon the nucleic acid or polypeptide of the invention.For example, 12 mg of JNJ-42756493 can be orally administered daily.JNJ-42756493 can be administered in seven to ten day repeating cycles(i.e. administration of JNJ-42756493 for seven to ten days, followed byno administration of JNJ-42756493 for seven to ten days). These amountscan be readily determined by a skilled artisan. Any of the therapeuticapplications described herein can be applied to any subject in need ofsuch therapy, including, for example, a mammal such as a dog, a cat, acow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human.

Pharmaceutical compositions for use in accordance with the invention canbe formulated in conventional manner using one or more physiologicallyacceptable carriers or excipients. The therapeutic compositions of theinvention can be formulated for a variety of routes of administration,including systemic and topical or localized administration. Techniquesand formulations generally can be found in Remmington's PharmaceuticalSciences, Meade Publishing Co., Easton, Pa. (20^(th) Ed., 2000), theentire disclosure of which is herein incorporated by reference. Forsystemic administration, an injection is useful, includingintramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the therapeutic compositions of the invention can beformulated in liquid solutions, for example in physiologicallycompatible buffers such as Hank's solution or Ringer's solution. Inaddition, the therapeutic compositions can be formulated in solid formand redissolved or suspended immediately prior to use. Lyophilized formsare also included. Pharmaceutical compositions of the present inventionare characterized as being at least sterile and pyrogen-free. Thesepharmaceutical formulations include formulations for human andveterinary use.

According to the invention, a pharmaceutically acceptable carrier cancomprise any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Any conventional media or agent that is compatible with theactive compound can be used. Supplementary active compounds can also beincorporated into the compositions.

A pharmaceutical composition containing FGFR fusion molecule inhibitorcan be administered in conjunction with a pharmaceutically acceptablecarrier, for any of the therapeutic effects discussed herein. Suchpharmaceutical compositions can comprise, for example antibodiesdirected to a FGFR fusion molecule, or a variant thereof, or antagonistsof a FGFR fusion molecule, or JNJ-42756493. The compositions can beadministered alone or in combination with at least one other agent, suchas a stabilizing compound, which can be administered in any sterile,biocompatible pharmaceutical carrier including, but not limited to,saline, buffered saline, dextrose, and water. The compositions can beadministered to a patient alone, or in combination with other agents,drugs or hormones.

Sterile injectable solutions can be prepared by incorporating the FGFRfusion molecule inhibitor (e.g., a polypeptide or antibody) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated herein.In the case of sterile powders for the preparation of sterile injectablesolutions, examples of useful preparation methods are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

In some embodiments, the FGFR fusion molecule inhibitor can be appliedvia transdermal delivery systems, which slowly releases the activecompound for percutaneous absorption. Permeation enhancers can be usedto facilitate transdermal penetration of the active factors in theconditioned media. Transdermal patches are described in for example,U.S. Pat. No. 5,407,713; U.S. Pat. No. 5,352,456; U.S. Pat. No.5,332,213; U.S. Pat. No. 5,336,168; U.S. Pat. No. 5,290,561; U.S. Pat.No. 5,254,346; U.S. Pat. No. 5,164,189; U.S. Pat. No. 5,163,899; U.S.Pat. No. 5,088,977; U.S. Pat. No. 5,087,240; U.S. Pat. No. 5,008,110;and U.S. Pat. No. 4,921,475.

“Subcutaneous” administration can refer to administration just beneaththe skin (i.e., beneath the dermis). Generally, the subcutaneous tissueis a layer of fat and connective tissue that houses larger blood vesselsand nerves. The size of this layer varies throughout the body and fromperson to person. The interface between the subcutaneous and musclelayers can be encompassed by subcutaneous administration. This mode ofadministration can be feasible where the subcutaneous layer issufficiently thin so that the factors present in the compositions canmigrate or diffuse from the locus of administration. Thus, whereintradermal administration is utilized, the bolus of compositionadministered is localized proximate to the subcutaneous layer.

Administration of the cell aggregates (such as DP or DS aggregates) isnot restricted to a single route, but can encompass administration bymultiple routes. For instance, exemplary administrations by multipleroutes include, among others, a combination of intradermal andintramuscular administration, or intradermal and subcutaneousadministration. Multiple administrations can be sequential orconcurrent. Other modes of application by multiple routes will beapparent to the skilled artisan.

In other embodiments, this implantation method will be a one-timetreatment for some subjects. In further embodiments of the invention,multiple cell therapy implantations will be required. In someembodiments, the cells used for implantation will generally besubject-specific genetically engineered cells. In another embodiment,cells obtained from a different species or another individual of thesame species can be used. Thus, using such cells can requireadministering an immunosuppressant to prevent rejection of the implantedcells. Such methods have also been described in U.S. Pat. No. 7,419,661and PCT application publication WO 2001/32840, and are herebyincorporated by reference.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation or ingestion), transdermal(topical), transmucosal, and rectal administration. For example,JNJ-42756493 can be orally administered. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, apharmaceutically acceptable polyol like glycerol, propylene glycol,liquid polyethylene glycol, and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it can be useful to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating theinhibitor (e.g., a polypeptide or antibody or small molecule) of theinvention in the required amount in an appropriate solvent with one or acombination of ingredients enumerated herein, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated herein. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of useful preparation methods arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions (e.g. of JNJ-42756493) generally include an inertdiluent or an edible carrier. They can be enclosed in gelatin capsulesor compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier and subsequently swallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

In some embodiments, the effective amount of the administered FGFRfusion molecule inhibitor (e.g. JNJ-42756493) is at least about 0.0001μg/kg body weight, at least about 0.00025 μg/kg body weight, at leastabout 0.0005 μg/kg body weight, at least about 0.00075 μg/kg bodyweight, at least about 0.001 μg/kg body weight, at least about 0.0025μg/kg body weight, at least about 0.005 μg/kg body weight, at leastabout 0.0075 μg/kg body weight, at least about 0.01 μg/kg body weight,at least about 0.025 μg/kg body weight, at least about 0.05 μg/kg bodyweight, at least about 0.075 μg/kg body weight, at least about 0.1 μg/kgbody weight, at least about 0.25 μg/kg body weight, at least about 0.5μg/kg body weight, at least about 0.75 μg/kg body weight, at least about1 μg/kg body weight, at least about 5 μg/kg body weight, at least about10 μg/kg body weight, at least about 25 μg/kg body weight, at leastabout 50 μg/kg body weight, at least about 75 μg/kg body weight, atleast about 100 μg/kg body weight, at least about 150 μg/kg body weight,at least about 200 μg/kg body weight, at least about 250 μg/kg bodyweight, at least about 300 μg/kg body weight, at least about 350 μg/kgbody weight, at least about 400 μg/kg body weight, at least about 450μg/kg body weight, at least about 500 μg/kg body weight, at least about550 μg/kg body weight, at least about 600 μg/kg body weight, at leastabout 650 μg/kg body weight, at least about 700 μg/kg body weight, atleast about 750 μg/kg body weight, at least about 800 μg/kg body weight,at least about 850 μg/kg body weight, at least about 900 μg/kg bodyweight, at least about 950 μg/kg body weight, at least about 1,000 μg/kgbody weight, at least about 2,000 μg/kg body weight, at least about3,000 μg/kg body weight, at least about 4,000 μg/kg body weight, atleast about 5,000 μg/kg body weight, at least about 6,000 μg/kg bodyweight, at least about 7,000 μg/kg body weight, at least about 8,000μg/kg body weight, at least about 9,500 μg/kg body weight, or at leastabout 10,000 μg/kg body weight.

In some embodiments, the effective amount of the administered FGFRfusion molecule inhibitor (e.g. JNJ-42756493) is at least about 1 mg, 2mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 14mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Exemplary methods and materialsare described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention.

All publications and other references mentioned herein are incorporatedby reference in their entirety, as if each individual publication orreference were specifically and individually indicated to beincorporated by reference. Publications and references cited herein arenot admitted to be prior art.

EXAMPLES

Examples are provided below to facilitate a more complete understandingof the invention. The following examples illustrate the exemplary modesof making and practicing the invention. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only, since alternativemethods can be utilized to obtain similar results.

The invention is further illustrated in Singh et al., Science (2012),337(6099):1231-5 (including the accompanying Supplementary Information).The entire contents of Singh et al., Science (2012), 337(6099):1231-5,including the accompanying “Supplementary Information,” is expresslyincorporated by reference. The invention is also further illustrated inDi Stefano et al., “Detection, characterization and inhibition ofFGFR-TACC fusions in IDH wild type glioma” Clin. Cancer Res. (2015), theentire contents of which are expressly incorporated by reference.

Example 1 Transforming and Recurrent Fusions of FGFR and TACC Gene inGlioblastoma

The history of successful targeted therapy of cancer largely coincideswith the inactivation of recurrent, oncogenic and addicting gene fusionsin hematological malignancies and recently in some types of epithelialcancer. Glioblastoma multiforme (GBM) is among the most lethal forms ofhuman cancer. Here, an integrated gene fusion discovery pipeline wasdeveloped for the detection of in-frame fused transcripts from RNA-seqand genomic fusions from whole exome sequences. The application of thepipeline to human GBM unraveled recurrent chromosomal translocations,which fuse in-frame the tyrosine kinase domain of FGFR genes (FGFR1 orFGFR3) to the TACC domain of TACC1 or TACC3, respectively. The frequencyof FGFR-TACC fusions is 3 of 97 GBM (3.1%). The FGFR-TACC fusion proteindisplays strong oncogenic activity when introduced into astrocytes ortransduced by lentivirus-mediated stereotactic delivery to the adultmouse brain. The FGFR-TACC fusion protein mis-localizes over the mitoticspindle pole, has constitutive tyrosine kinase activity and dysregulatesthe mitotic cycle with delayed mitotic progression. The impaired mitoticfidelity triggers chromatid cohesion defects, defective spindlecheckpoint activation, chromosomal mis-segregation, and rampantaneuploidy. Inhibition of FGFR kinase corrects the aneuploidy and oraladministration of a specific FGFR tyrosine kinase inhibitor underclinical investigation arrests tumor growth and prolongs survival ofmice harboring intracranial FGFR3-TACC3-initiated glioma. FGFR-TACCfusions identify a subset of GBM patients who may benefit from targetedinhibition of the tyrosine kinase activity of FGFR.

Glioblastoma multiforme (GBM) is among the most difficult forms ofcancer to treat in humans (Ohgaki and Kleihues, 2005). So far, thetargeted therapeutic approaches that have been tested againstpotentially important oncogenic drivers in GBM have met limited success(Lo, 2010; Reardon et al., 2010; van den Bent et al., 2009). Recurrentchromosomal translocations leading to production of oncogenic fusionproteins are viewed as initiating and addicting events in thepathogenesis of human cancer, thus providing the most desirablemolecular targets for cancer therapy (Ablain et al., 2011; Mitelman etal., 2007). Chromosomal rearrangements resulting in recurrent andoncogenic gene fusions are hallmarks of hematological malignancies andrecently they have also been uncovered in subsets of solid tumors(breast, prostate, lung and colorectal carcinoma), but they have notbeen found in GBM (Bass et al., 2011; Prensner and Chinnaiyan, 2009).Important and successful targeted therapeutic interventions for patientswhose tumors carry these rearrangements have stemmed from the discoveryof functional gene fusions, especially when the translocations involvekinase-coding genes (BCR-ABL, EML4-ALK) (Druker, 2009; Gerber and Minna,2010).

A hallmark of GBM is rampant chromosomal instability (CIN), which leadsto aneuploidy (Furnari et al., 2007). CIN and aneuploidy are earlyevents in the pathogenesis of cancer (Cahill et al., 1999). It has beensuggested that genetic alterations targeting mitotic fidelity might beresponsible for mis-segregation of chromosomes during mitosis, resultingin aneuploidy (Gordon et al., 2012; Solomon et al., 2011). Here, thefirst cases of recurrent and oncogenic gene fusions in human GBM aredescribed. The resulting fusion protein localizes to mitotic cells,disrupts the normal control of chromosome segregation and inducesaneuploidy. A therapeutic strategy with FGFR tyrosine kinase inhibitorsis also reported for the targeted therapy of GBM patients harboringthese chromosomal rearrangements.

Identification of Recurrent Fusions of FGFR and TACC Genes.

To identify genomic rearrangements in GBM that generate functionalfusion proteins and are recurrent, gene pairs discovered as in-framefused transcripts from the analysis of massively parallel, paired-endsequencing of expressed transcripts (RNA-seq) would also emerge as fusedgene pairs from the genomic analysis of human GBM. Towards this aim, twocomplementary gene fusion discovery methods were devised and wereapplied to two GBM cohorts. The first, TX-Fuse, is an algorithm for thediscovery of candidate fusion transcripts from RNA-seq (FIG. 8). Thesecond, Exome-Fuse, detects fusion genes from whole exome DNA sequences(FIG. 8). As first step for the detection of fused transcripts, RNA-seqdata was generated from short-term cultures of glioma stem-like cells(GSCs) freshly isolated from nine patients carrying primary GBM. Theculture of primary GBM tumors under serum-free conditions selects cellsthat retain phenotypes and genotypes closely mirroring primary tumorprofiles as compared to serum-cultured glioma cell lines that havelargely lost their developmental identities (Lee et al., 2006).Therefore, without being bound by theory, if glioma cells carry genefusions causally responsible for the most aggressive hallmarks of GBM,they should be selected in GSCs. RNA-seq generated an average of 60.3million paired reads for each GSC culture, of which over 80% were mappedto the reference transcriptome and genome. TX-Fuse detects two mainsources of evidence: split reads and split inserts (see ExperimentalProcedures). The application of TX-Fuse to the RNA-seq dataset from nineGSCs led to the discovery of five candidate rearrangements (all of whichwere intrachromosomal) that give rise to in-frame fusion transcripts(Table 1).

TABLE 1 Predicted in-frame fusion proteins from RNA-Seq of nine GSCs #Split # Split Ref Ref Tx Tx Inserts Reads Sample Gene1 Gene2 Seq1 Seq2Pos1 Pos2 294 76 GSC- FGFR3 TACC3 NM_000142 NM_006342 2530 1751 1123 3754 GSC- POLR2A WRAP53 NM_000937 NM_001143990 479 798 0114 7 48 GSC-CAPZB UBR4 NM_001206540 NM_020765 228 12111 0114 8 29 GSC- ST8SIA4 PAMNM_005668 NM_000919 1125 730 0517 6 17 GSC- PIGU NCOA6 NM_080476NM_014071 729 6471 0308 1 6 GSC- IFNAR2 IL10RB NM_000874 NM_000628 1083149 0127 #Split #Split Inserts Reads Sample Chr1 Strand1 hg19_GenPos1Chr2 Strand2 hg19_GenPos2 294 76 GSC- 4 + 1808842 4 + 1737004 1123 37 54GSC- 17 + 7399259 17 + 7604059 0114 7 48 GSC- 1 − 19712098 1 − 194334400114 8 29 GSC- 5 − 100147809 5 + 102260661 0517 6 17 GSC- 20 − 3320391420 − 33303130 0308 1 6 GSC- 21 + 34632901 21 + 34640699 0127

Next, genomic rearrangements leading to gene fusions were identified inGBM by applying Exome-Fuse to a dataset of paired-end exome DNAsequences from 84 GBM samples from TCGA (Table 2).

This analysis detected 147 paired gene fusions, thus producing anaverage of 1.75 gene fusion events per tumor (Table 3).

The FGFR and TACC families of genes were markedly enriched among thoserecurrently involved in genomic fusions, with eight tumors harboringFGFR rearrangements and seven tumors harboring fusions that implicateTACC genes (FIG. 1A). The comparative analysis of the TX-Fuse andExon-Fuse outputs revealed that FGFR3-TACC3 was the only fusion pairidentified as either an in-frame transcript by TX-Fuse and genomicfusions by Exome-Fuse (Tables 1, 2 and 3).

Table 2 shows fusion breakpoint information of recurrent gene fusionsidentified by Exome-fuse analysis of 84 GBM from TCGA. As multiplejunctions may exist in each fusion candidate, information for allbreakpoints is displayed. Column definitions include: sample=TCGA sampleID, virtForSplitReads/virtRevSplitReads/virtTotSplitReads=#forward/reverse/total split reads, splitInserts=# split inserts,dirA/dirB=forward (1) or reverse (0) direction of split read portionmapping to gene A/B, dirAB_matepair=direction of mate pair of splitread, cosmicA+B=# recorded mutations of gene A+B in COSMIC.

TABLE 2 Fusion breakpoint information of recurrent gene fusionsidentified by Exome-fuse analysis of 84 GBM from TCGA. cosmic SamplevlrtForSplitReads vlrtRevSplitReads vlrtForSplitReads splitinserts geneAchrA senseA posA geneB chrB senseB posB chrA chrB dirAB_matepair A + BTCGA-06-6390 10 9 19 8 FGFR3 chr4 + 1778521 TACC3 chr4 + 1708787 1 1 02803 TCGA-12-0826 5 6 11 5 FGFR3 chr4 + 1778502 TACC3 chr4 + 1707185 0 01 2803 TCGA-19-5958 3 0 3 2 FGFR3 chr4 + 1778539 TACC3 chr4 + 1707203 01 1 2803 TCGA-27-1835 11 1 12 4 FGFR3 chr4 + 1778595 TACC3 chr4 +1709397 0 0 1 2803 TCGA-12-0820 7 2 9 4 FGFR3 chr4 + 1779184 PRKG2 chr4− 82338347 1 1 0 2805 TCGA-12-1088 3 1 4 4 ABL1 chr9 + 132597569TNFRSF10B chr8 − 22936252 0 0 1 892 TCGA-06-1802 7 1 8 8 ADAM12 chr10 −127698245 PTPRD chr9 − 8596127 0 0 1 54 TCGA-06-1801 7 0 7 5 HIP1 chr7 −75010010 PTPRD chr9 − 9387093 1 0 0 52 TCGA-12-1088 3 0 3 3 KIDINS220chr2 − 8886300 PPP1R3A chr7 − 113305567 0 0 1 45 TCGA-12-1088 37 1 38 10KIDINS220 chr2 − 8887075 PPP1R3A chr7 − 113305191 1 1 0 45 TCGA-32-24912 17 19 6 ODZ1 chrX − 123342503 STAG2 chrX + 123019118 0 1 0 36TCGA-32-2491 11 1 12 10 ODZ1 chrX − 123526882 SASH3 chrX + 128749198 1 00 34 TCGA-12-0829 24 0 24 13 LRRK2 chr12 + 39032542 VSNL1 chr2 +17630556 1 1 0 32 TCGA-12-0829 25 1 26 13 LRRK2 chr12 + 38975444 VSNL1chr2 + 17639377 1 0 0 32 TCGA-12-0829 87 16 103 58 LRRK2 chr12 +38975652 VSNL1 chr2 + 17639552 0 1 1 32 TCGA-19-0957 3 2 5 6 NUDT19chr19 + 37891921 ODZ1 chrX − 123925223 1 0 0 32 TCGA-12-1088 12 1 13 5GLI3 chr7 − 42031380 RIMBP2 chr12 − 129517282 1 0 0 31 TCGA-12-1088 5 05 1 GLI3 chr7 − 42031574 RIMBP2 chr12 − 129517455 0 1 1 31 TCGA-12-108910 0 10 5 AHNAK chr11 − 62056459 C21orf29 chr21 − 44923276 0 1 1 30TCGA-06-1801 27 1 28 12 CROCC chr1 + 17171362 CSMD2 chr1 − 34381139 1 00 29 TCGA-12-1089 12 1 13 6 CLK3 chr15 + 72705401 LRP1 chr12 + 558800020 1 1 28 TCGA-12-1089 14 2 16 8 CLK3 chr15 + 72705248 LRP1 chr12 +55879646 1 0 0 28 TCGA-12-1089 42 5 47 24 LAMA2 chr6 + 129836071 PDE10Achr6 − 165858426 1 0 0 28 TCGA-06-1802 48 9 57 27 LAMA2 chr6 + 129483265SEC14L3 chr22 − 29193005 1 1 0 27 TCGA-19-0957 4 16 20 6 CSMD2 chr1 −34115076 MDH2 chr7 + 75525221 0 0 1 27 TCGA-06-1801 21 1 22 4 FAM192Achr16 − 55757701 LRP1 chr12 + 55858598 0 0 1 26 TCGA-12-1089 27 0 27 2FGFR4 chr5 + 176447670 LILRB1 chr19 + 59840807 1 0 0 25 TCGA-19-0957 0 11 4 EML1 chr14 + 99349006 NRXN3 chr14 + 79233969 1 0 0 24 TCGA-06-180119 133 152 51 NHSL2 chrX + 71082676 TAF1 chrX + 70520522 1 0 0 22TCGA-06-1801 51 3 54 8 NHSL2 chrX + 71083319 TAF1 chrX + 70521607 1 0 122 TCGA-12-1089 9 0 9 4 CACNA1C chr12 + 2325330 ITGAV chr2 + 187195411 00 1 22 TCGA-19-0957 8 1 9 6 CDH11 chr16 − 63579650 RERE chr1 − 8588774 00 1 22 TCGA-12-0829 12 3 15 4 ENTPD2 chr9 − 139062591 FREM2 chr13 +38318644 1 1 0 21 TCGA-12-0829 2 3 5 1 EFS chr14 − 22896776 NRXN3 ch14 +78678529 1 0 1 21 TCGA-12-0829 56 6 62 14 DIS3L chr15 + 64377566 GLI3chr7 − 42032535 1 0 TCGA-12-0829 8 2 10 3 EFS chr14 − 22896431 NRXN3chr14 + 78678139 1 0 TCGA-12-0829 9 65 74 37 DIS3L chr15 + 64377398 GLI3chr7 − 42032341 1 0 TCGA-27-1835 14 0 14 4 FAM19A2 chr12 − 60707200 GLI1chr12 + 56146523 0 0 TCGA-06-1801 20 0 20 2 FREM2 chr13 + 38163882 RALYLchr8 + 85785432 1 1 TCGA-12-0827 2 0 2 2 ABCC12 chr16 − 46722685 FGFR4chr5 + 176457194 1 1 TCGA-12-0829 35 0 35 7 ANXA7 chr10 − 74808655CACNA1C chr12 + 2458351 1 1 TCGA-06-2559 60 37 97 1 PLEKHM3 chr2 −208426920 PTPRS chr19 − 5222592 0 0 TCGA-12-1088 2 0 2 2 PLCL1 chr2 +198630224 TACC2 chr10 + 123987513 1 1 TCGA-06-1801 10 0 10 4 FGFR4chr5 + 176450528 WISP2 chr20 + 42782576 0 1 TCGA-06-1802 15 0 15 2 PDHA2chr4 + 96980717 PDZRN4 chr12 + 39959553 0 1 TCGA-06-1802 4 0 4 2 PDHA2chr4 + 96980509 PDZRN4 chr12 + 39959384 1 0 TCGA-06-6390 53 0 53 18GPR182 chr12 + 55675639 PDZRN4 chr12 + 39957003 1 0 TCGA-12-0829 1121252 1373 602 ADCY8 chr8 − 131886108 SSX3 chrX − 48091929 0 0TCGA-12-0829 14 8 22 3 ADCY8 chr8 − 131886506 SSX3 chrX − 48091719 1 1TCGA-12-0829 9 42 51 18 ADAM12 chr10 − 127733231 DAPK1 chr9 + 89454764 01 TCGA-12-3653 22 0 22 10 JOSD2 chr19 − 55705579 PTPRS chr19 − 5245999 01 TCGA-12-0829 100 0 100 20 COL14A1 chr8 + 121370990 MMP12 chr11 −102242881 1 0 TCGA-12-0829 152 0 152 24 COL14A1 chr8 + 121371195 MMP12chr11 − 102242953 0 1 TCGA-06-1802 11 47 58 19 MUSK chr9 + 112509906SYNPO2 chr4 + 120172123 0 0 TCGA-06-1805 6 4 10 6 COL14A1 chr8 +121332080 NCRNA0015 chr21 − 18174873 1 1 TCGA-12-0822 37 0 37 3 C7orf44chr7 − 43683128 TACC2 chr10 + 123835337 1 0 TCGA-12-0829 0 2 2 365 GSTA3chr6 − 52878492 TACC2 chr10 + 123884543 0 1 TCGA-12-0829 124 16 140 51GSTA3 chr6 − 52878680 TACC2 chr10 + 123884705 0 1 TCGA-12-0829 21 7 2810 HIP1 chr7 − 75022909 MASP1 chr3 − 188452372 0 0 TCGA-12-0829 268 123391 242 HIP1 chr7 − 75022741 MASP1 chr3 − 188452581 1 1 TCGA-12-0829 36641 677 365 GSTA3 chr6 − 52878496 TACC2 chr10 + 123884531 0 1TCGA-12-1088 10 1 11 3 CAMTA1 chr1 + 7710762 TMPRSS3 chr21 − 42665918 01 TCGA-12-1088 65 0 65 6 ADCY10 chr1 − 166139873 DUSP27 chr1 + 1653515550 0 TCGA-12-1088 8 1 9 4 CAMTA1 chr1 + 7714539 TMPRSS3 chr21 − 426660441 0 TCGA-27-1835 83 1 84 22 CMYA5 chr5 + 79120729 SRRM1 chr1 + 248708990 0 TCGA-06-1801 0 43 43 31 CAMTA1 chr1 + 7264935 GDPD2 chrX + 695637590 1 TCGA-06-1801 13 41 54 31 CAMTA1 chr1 + 7265429 GDPD2 chrX + 695634311 0 TCGA-06-1801 24 66 90 61 CAMTA1 chr1 + 7265556 GDPD2 chrX + 695637620 1 TCGA-12-0829 2 0 2 3 CCDC147 chr10 + 106165013 ISX chr22 + 337957080 1 TCGA-12-1088 7 1 8 5 CMYA5 chr5 + 79045621 STK24 chr13 − 97969547 10 TCGA-06-1801 7 1 8 4 DEPDC5 chr22 + 30619774 ROBO1 chr3 − 79802538 0 1TCGA-12-0820 110 20 130 23 ABCA13 chr7 + 48597322 NHSL2 chrX + 710775471 0 TCGA-12-0820 29 4 33 3 ABCA13 chr7 + 48597477 NHSL2 chrX + 710776900 1 TCGA-12-0829 46 2 48 4 LIN9 chr1 − 224536835 NCOR1 chr17 − 158835850 0 TCGA-12-3644 3 0 3 1 EFHC1 chr6 + 52432073 LRBA chr4 − 151418615 1 0TCGA-12-3644 3 10 13 3 EFHC1 chr6 + 52431890 LRBA chr4 − 151418438 1 0TCGA-19-5958 6 6 12 7 DEPDC5 chr22 + 30504095 SLC5A4 chr22 − 30974671 01 TCGA-06-1801 4 4 8 5 KCND3 chr1 − 112227957 LY75 chr2 − 160443238 1 1TCGA-12-0820 26 1 27 2 BBX chr3 + 108997451 CUL3 chr2 − 225108623 0 0TCGA-12-0828 8 67 75 31 ADCY2 chr5 + 7558840 SDAD1 chr4 − 77096208 1 1TCGA-12-0829 13 21 34 16 AGBL4 chr1 − 48902776 NUP188 chr9 + 130808425 00 TCGA-12-0829 64 308 372 197 EYS chr6 − 64513356 IL1RN chr2 + 1136037120 1 TCGA-12-0829 7 25 32 11 AGBL4 chr1 − 48902600 NUP188 chr9 +130808628 1 1 TCGA-12-0829 9 0 9 1 LRBA chr4 − 151790893 PSEN1 chr14 +72707609 1 0 TCGA-12-1093 65 4 69 21 OSBPL10 chr3 − 31687272 TRAPPC9chr8 − 140828099 1 1 TCGA-12-1600 9 0 9 5 5-Sep chr22 + 18088018 NCOR1chr17 − 15915170 0 1 TCGA-19-0957 18 1 19 7 ADCY10 chr1 − 166060645 AKT3chr1 − 241743142 0 1 TCGA-19-0957 34 2 36 11 ADCY10 chr1 − 166060502AKT3 chr1 − 241742588 1 0 TCGA-12-0822 0 1 1 16 ITGB2 chr21 − 45147805SH3RF3 chr2 + 109430489 0 1 TCGA-12-0822 6 2 8 1 ITGB2 chr21 − 45147994SH3RF3 chr2 + 109430669 0 1 TCGA-12-0827 25 3 28 8 CUL3 chr2 − 225126210LY75 chr2 − 160455052 1 0 TCGA-12-0828 7 2 9 4 FH chr1 − 239743589SRGAP1 chr12 + 62723692 0 1 TCGA-12-0829 24 0 24 9 ITGA9 chr3 + 37712050SNX5 chr20 − 17885523 0 1 TCGA-12-1089 17 2 19 5 ABCC1 chr16 + 16077635RNF216 chr7 − 5692038 1 1 TCGA-12-1089 6 0 6 8 CAMSAP1 chr9 − 137867066NCF2 chr1 − 181799323 1 0 TCGA-19-0957 16 0 16 4 CCDC147 chr10 +106114657 STK4 chr20 + 43111359 0 0 TCGA-06-1801 5 33 38 18 AP4S1chr14 + 30611930 EYS chr6 − 64770011 1 0 TCGA-06-1805 3 14 17 9 CUL3chr2 − 225064315 SLC44A2 chr19 + 10608393 1 0 TCGA-12-0829 14 27 41 23ADCY2 chr5 + 7798046 C14orf174 chr14 + 76914809 1 0 TCGA-12-0829 59 7 6618 NR3C1 chr5 − 142760085 SORCS2 chr4 + 7354165 0 0 TCGA-12-0829 9 40 4928 ADCY2 chr5 + 7798641 C14orf174 chr14 + 76915034 0 1 TCGA-12-1093 20 020 4 GAPVD1 chr9 + 127104266 MAPKAP1 chr9 − 127490362 1 0 TCGA-12-1600 70 7 4 CILP chr15 − 63283865 PARP16 chr15 − 63350048 0 1 1 10TCGA-19-0957 13 3 16 6 AQP2 chr12 + 48635567 CDH4 chr20 + 59413648 0 1 110 TCGA-19-0957 6 0 6 1 AQP2 chr12 + 48635406 CDH4 chr20 + 59413468 1 00 10 TCGA-06-0166 2 0 2 3 CCDC158 chr4 − 77541796 SNX5 chr20 − 178853460 1 1 9 TCGA-06-1802 30 0 30 9 RANBP2 chr2 + 108758804 SATB2 chr2 −199895572 0 1 1 9 TCGA-06-1805 4 0 4 3 C2CD3 chr11 − 73430819 XRRA1chr11 − 74309669 1 1 0 9 TCGA-06-1805 6 1 7 5 NEUROG1 chr5 − 134898853PRKCH chr14 + 51027580 1 1 0 9 TCGA-12-0820 27 0 27 2 RANBP2 chr2 +108749908 TTC27 chr2 + 32839367 0 1 1 9 TCGA-12-0820 58 7 65 15 RANBP2chr2 + 108749412 TTC27 chr2 + 32837790 1 0 0 9 TCGA-12-0829 6 128 134 35C2CD3 chr11 − 73529639 CAPZB chr1 − 19556435 0 0 1 9 TCGA-12-0829 84 443527 227 C2CD3 chr11 − 73529293 CAPZB chr1 − 19556627 1 1 0 9TCGA-12-1088 10 0 10 2 PACSIN1 chr6 + 34589431 TNC chr9 − 116884742 0 10 9 TCGA-12-1088 12 0 12 2 PACSIN1 chr6 + 34589619 TNC chr9 − 1168849581 0 1 9 TCGA-19-0957 34 19 53 17 PRKCH chr14 + 61032978 ZFAND3 chr6 +38228111 1 0 1 9 TCGA-19-0957 7 1 8 7 MAPKAP1 chr9 − 127348507 SLC9A1chr1 − 27302334 0 1 0 9 TCGA-19-0957 8 39 47 21 PRKCH chr14 + 61032774ZFAND3 chr6 + 38227949 1 0 0 9 TCGA-06-1801 5 11 16 4 MAOA chrX +43486192 SH3RF3 chr2 + 109237058 1 0 1 8 TCGA-06-1802 10 12 22 12 DNM1Lchr12 + 32736794 SYNPO2 chr4 + 120172271 0 1 1 8 TCGA-06-1802 18 42 6024 MUC4 chr3 − 196982875 SMOC2 chr6 + 168676813 1 0 1 8 TCGA-12-0829 6 06 6 ATXN1 chr6 − 16669201 CACNA1G chr17 + 46004995 0 0 1 8 TCGA-12-08297 0 7 3 ATP6V0D2 chr8 + 87186716 RERE chr1 − 8336574 1 1 0 8TCGA-12-1088 11 1 12 6 BCAS3 chr17 + 56321892 CACNA1G chr17 + 46010698 11 0 8 TCGA-12-1088 15 2 17 5 ABCC1 chr16 + 16135771 AGBL4 chr1 −49315120 1 0 0 8 TCGA-12-1088 17 3 20 4 MST1R chr3 − 49910627 WDFY1 chr2− 224512774 1 0 0 8 TCGA-12-1088 4 0 4 2 FBXL4 chr6 − 99431443 SYNPO2chr4 + 120172560 0 0 1 8 TCGA-12-1092 39 4 43 12 CNTN2 chr1 + 203302926DNAJC6 chr1 + 65591195 0 0 1 8 TCGA-12-1598 4 0 4 5 MPP1 chrX −153673715 SRGAP1 chr12 + 62777947 0 0 1 8 TCGA-19-1786 5 19 24 7 ATP5Bchr12 − 55320148 USP48 chr1 − 21920103 1 0 1 8 TCGA-19-2621 21 0 21 3BCAS3 chr17 + 56731673 TTYH1 chr19 + 59638801 1 1 0 8 TCGA-06-1801 15 015 9 C15orf23 chr15 + 38469150 DMD chrX − 32092185 0 1 1 7 TCGA-06-18056 3 9 5 FAM19A2 chr12 − 60547321 POLM chr7 − 44082653 0 1 0 7TCGA-12-0829 13 84 97 44 ATP5B chr12 − 55318484 PRC1 chr15 − 89330475 10 1 7 TCGA-12-0829 158 207 365 44 ATP5B chr12 − 55320850 PRC1 chr15 −89334458 1 1 0 7 TCGA-12-0829 2 1 3 2 DDI2 chr1 + 15825507 KIDINS220chr2 − 8805399 0 1 0 7 TCGA-12-0829 25 6 31 44 ATP5B chr12 − 55321832PRC1 chr15 − 89335627 1 0 1 7 TCGA-12-0829 34 21 55 44 ATP5B chr12 −55321200 PRC1 chr15 − 89335044 0 1 0 7 TCGA-12-0829 44 6 50 4 ABCC6chr16 − 16204784 SUMF1 chr3 − 4470138 1 0 0 7 TCGA-12-0829 53 28 81 35DDI2 chr1 + 15825941 KIDINS220 chr2 − 8805580 1 0 1 7 TCGA-12-0829 9 0 95 DMD chrX − 32013100 N4BP2L2 chr13 − 32008512 0 0 1 7 TCGA-12-1092 9 09 4 LRRC48 chr19 − 55754780 NR3C1 chr5 − 142660156 0 1 0 7 TCGA-19-26213 22 25 11 PCDH12 chr5 − 141309153 SLC36A2 chr5 − 150679274 1 1 0 7TCGA-06-1802 8 4 12 6 BAHD1 chr15 + 38539023 OSBPL10 chr3 − 31729622 0 11 6 TCGA-12-0828 11 0 11 1 PLOD3 chr7 − 100646340 VSNL1 chr2 + 176386181 1 0 6 TCGA-12-0828 40 9 49 20 PLOD3 chr7 − 100646511 VSNL1 chr2 +17637955 0 0 1 6 TCGA-12-0829 16 2 18 9 C21orf29 chr21 − 44922864 MYT1chr20 + 62300829 0 0 1 6 TCGA-12-0829 196 0 196 37 IGFBP3 chr7 −45922866 SMOC2 chr6 + 168722450 0 0 1 6 TCGA-12-0829 5 1 6 1 FAM168Achr11 − 72839771 NCF2 chr1 − 181826115 1 0 1 6 TCGA-12-0829 5 18 23 9FAM168A chr11 − 72839534 NCF2 chr1 − 181825930 1 0 0 6 TCGA-12-1089 20 020 2 SLC44A2 chr19 + 10602997 XRCC4 chr5 + 82430803 1 0 0 6 TCGA-19-095717 1 18 4 PAX3 chr2 − 222778052 WDFY1 chr2 − 224453159 1 0 1 6TCGA-06-1801 5 0 5 1 CAP2 chr6 + 17571234 DNAJC6 chr1 + 65602700 1 0 0 5TCGA-06-1801 6 32 38 15 CAP2 chr6 + 17571666 DNAJC6 chr1 + 65603089 0 11 5 TCGA-06-1805 3 0 3 8 PLCL1 chr2 + 198578552 SURF6 chr9 − 135188818 01 0 5 TCGA-06-1805 7 2 9 4 PLCL1 chr2 + 198578671 SURF6 chr9 − 1351892941 0 1 5 TCGA-12-0822 17 4 21 4 TAAR6 chr6 + 132933266 TTYH1 chr19 +59629451 0 1 1 5 TCGA-12-0828 17 0 17 8 AQP2 chr12 + 48634800 ECE1 chr1− 21515240 0 1 1 5 TCGA-12-0828 7 0 7 1 AQP2 chr12 + 48634610 ECE1 chr1− 21515033 1 0 0 5 TCGA-12-0829 12 0 12 7 CACNA1G chr17 + 46039372CNTNAP4 chr16 + 74873868 0 1 1 5 TCGA-19-0957 4 0 4 3 PCDH12 chr5 −141316624 SH3BP5 chr3 − 15315567 0 0 1 5 TCGA-19-0957 8 2 10 2 PCDH12chr5 − 141316405 SH3BP5 chr3 − 15315731 1 1 0 5 TCGA-06-1801 13 1 14 1ABCC6 chr16 − 16205051 CMTM7 chr3 + 32443880 0 1 1 4 TCGA-06-1801 33 740 4 ABCC6 chr16 − 16204860 CMTM7 chr3 + 32443722 1 0 0 4 TCGA-06-180510 3 13 6 AGBL4 chr1 − 49449813 NOX4 chr11 − 88714996 0 0 1 4TCGA-12-0829 11 0 11 4 FAM160A1 chr4 + 152595916 LY75 chr2 − 160440194 01 0 4 TCGA-12-0829 17 1 18 5 FAM160A1 chr4 + 152596097 LY75 chr2 −160440376 1 0 1 4 TCGA-12-0829 487 83 570 249 CORO7 chr16 − 4375428DYRK3 chr1 + 204876154 0 0 1 4 TCGA-12-1088 2 12 14 4 FAM172A chr5 −93052315 TRIOBP chr22 + 36427382 0 1 1 4 TCGA-06-1801 30 7 37 18 DEPDC7chr11 + 33003811 EIF2C2 chr8 − 141618836 1 0 0 3 TCGA-06-1801 40 28 6833 MAP7 chr6 − 136728609 SH3RF3 chr2 + 109392677 0 0 TCGA-12-1093 6 1521 8 CORO7 chr16 − 4398302 PLEK2 chr14 − 66934201 0 1 TCGA-12-3644 33 033 4 EDA chrX + 69073054 SSX3 chrX − 48094443 1 1 TCGA-12-3644 37 15 5217 C15orf33 chr15 − 47424122 PARP16 chr15 − 63350289 0 0 TCGA-19-1791 143 17 8 PSEN1 chr14 + 72748293 ZNF410 chr14 + 73431112 0 0 TCGA-06-180235 2 37 17 CELF2 chr10 + 11352537 PLA2G2F chr1 + 20348173 1 1TCGA-06-1802 63 26 89 25 CELF2 chr10 + 11352765 PLA2G2F chr1 + 203479970 0 TCGA-06-1802 8 2 10 8 LCLAT1 chr2 + 30535977 PACSIN1 chr6 + 345761951 0 TCGA-06-2562 6 0 6 4 SNTA1 chr20 − 31473415 TMEM80 chr11 + 689744 00 TCGA-12-0829 16 1 17 4 LASS6 chr2 + 169045211 NKAIN2 chr6 + 1250212520 0 TCGA-12-0829 7 0 7 2 LASS6 chr2 + 169045333 NKAIN2 chr6 + 1250210721 1 TCGA-14-0813 339 39 378 5 SNTA1 chr20 − 31481069 TMEM80 chr11 +686739 0 0 TCGA-12-0820 49 3 52 11 CAMKK1 chr17 − 3712344 FAM184B chr4 −17271273 0 1 TCGA-12-0826 8 18 26 13 CELF2 chr10 + 11406463 NME4 chr16 +389427 1 1 TCGA-12-1089 17 4 21 10 C6orf170 chr6 − 121478035 NKAIN2chr6 + 125083380 1 0 TCGA-12-1600 19 0 19 3 ATP6AP1L chr5 + 81649744FAM172A chr5 − 93336459 1 0 TCGA-12-1600 5 35 40 6 ATP6AP1L chr5 +81649902 FAM172A chr5 − 93336676 1 0 TCGA-19-1790 4 0 4 4 ARMC6 chr19 +19026932 FAM184B chr4 − 17391210 1 0 TCGA-06-1802 12 0 12 8 EIF2C2 chr8− 141648334 TNFRSF10B chr8 − 22940680 0 0 TCGA-14-0781 22 2 24 8FAM160A1 chr4 + 152584637 UNC93B1 chr11 − 67523253 1 0

TABLE 3 Recurrent gene fusion pairs from Exome- fuse analysis of 84 GBMfrom TCGA. Sample gene A gene B TCGA-12-0820 ABCA13 NHSL2 TCGA-12-1089ABCC1 RNF216 TCGA-12-1088 ABCC1 AGBL4 TCGA-12-0827 ABCC12 FGFR4TCGA-12-0829 ABCC6 SUMF1 TCGA-06-1801 ABCC6 CMTM7 TCGA-12-1088 ABL1TNFRSF10B TCGA-06-1802 ADAM12 PTPRD TCGA-12-0829 ADAM12 DAPK1TCGA-12-1088 ADCY10 DUSP27 TCGA-19-0957 ADCY10 AKT3 TCGA-12-0828 ADCY2SDAD1 TCGA-12-0829 ADCY2 C14orf174 TCGA-12-0829 ADCY8 SSX3 TCGA-12-0829AGBL4 NUP188 TCGA-06-1805 AGBL4 NOX4 TCGA-12-1089 AHNAK C21orf29TCGA-12-0829 ANXA7 CACNA1C TCGA-06-1801 AP4S1 EYS TCGA-12-0828 AQP2 ECE1TCGA-19-0957 AQP2 CDH4 TCGA-19-1790 ARMC6 FAM184B TCGA-19-1786 ATP5BUSP48 TCGA-12-0829 ATP5B PRC1 TCGA-12-1600 ATP6AP1L FAM172A TCGA-12-0829ATP6V0D2 RERE TCGA-12-0829 ATXN1 CACNA1G TCGA-06-1802 BAHD1 OSBPL10TCGA-12-0820 BBX CUL3 TCGA-19-2621 BCAS3 TTYH1 TCGA-12-1088 BCAS3CACNA1G TCGA-06-1801 C15orf23 DMD TCGA-12-3644 C15orf33 PARP16TCGA-12-0829 C21orf29 MYT1 TCGA-06-1805 C2CD3 XRRA1 TCGA-12-0829 C2CD3CAPZ8 TCGA-12-1089 C6orf170 NKAIN2 TCGA-12-0822 C7orf44 TACC2TCGA-12-1089 CACNA1C ITGAV TCGA-12-0829 CACNA1G CNTNAP4 TCGA-12-0820CAMKK1 FAM184B TCGA-12-1089 CAMSAP1 NCF2 TCGA-12-1088 CAMTA1 TMPRSS3TCGA-06-1801 CAMTA1 GDPD2 TCGA-06-1801 CAP2 DNAJC6 TCGA-19-0957 CCDC147STK4 TCGA-12-0829 CCDC147 ISX TCGA-06-0166 CCDC158 SNX5 TCGA-19-0957CDH11 RERE TCGA-06-1802 CELF2 PLA2G2F TCGA-12-0826 CELF2 NME4TCGA-12-1600 CILP PARP16 TCGA-12-1089 CLK3 LRP1 TCGA-12-1088 CMYA5 STK24TCGA-27-1835 CMYA5 SRRM1 TCGA-12-1092 CNTN2 DNAJC6 TCGA-06-1805 COL14A1NCRNA00157 TCGA-12-0829 COL14A1 MMP12 TCGA-12-1093 CORO7 PLEK2TCGA-12-0829 CORO7 DYRK3 TCGA-06-1801 CROCC CSMD2 TCGA-19-0957 CSMD2MDH2 TCGA-06-1805 CUL3 SLC44A2 TCGA-12-0827 CUL3 LY75 TCGA-12-0829 DDI2KIDINS220 TCGA-19-5958 DEPDC5 SLC5A4 TCGA-06-1801 DEPDC5 ROBO1TCGA-06-1801 DEPDC7 EIF2C2 TCGA-12-0829 DIS3L GLI3 TCGA-12-0829 DMDN4BP2L2 TCGA-06-1802 DNM1L SYNPO2 TCGA-12-3644 EDA SSX3 TCGA-12-3644EFHC1 LRBA TCGA-12-0829 EFS NRXN3 TCGA-06-1802 EIF2C2 TNFRSF10BTCGA-19-0957 EML1 NRXN3 TCGA-12-0829 ENTPD2 FREM2 TCGA-12-0829 EYS IL1RNTCGA-14-0781 FAM160A1 UNC93B1 TCGA-12-0829 FAM160A1 LY75

Table 3 above shows recurrent gene fusion pairs from Exome-fuse analysisof 84 GBM from TCGA. Fusion candidates have been nominated if they haveat least two split inserts and at least two split reads. To furtherfilter the list on recurrence, any fusion candidate was kept in whichone of the genes is involved in at least two fusions across differentsamples.

To experimentally validate the computational predictions that emergedfrom TX-Fuse, the PCR products spanning the fusion breakpoint weresequenced and validated each of the five in-frame fusion predictions(FIGS. 1 and 9). In FIG. 1B, the prediction is shown and in FIG. 1C, thecDNA sequence validation for the fusion with the highest read supportinvolving FGFR3 fused in-frame with TACC3 in GSC-1123 is shown. The sameFGFR3-TACC3 fusion transcript was also detected in the primary GBM-1123tumor specimen from which the GSC-1123 culture was established (FIG.1C). The amplified cDNA contained an open reading frame for a protein of1,048 amino acids resulting from the fusion of a FGFR3 amino-terminalportion of residues 1-758 with a TACC3 carboxy-terminal portion ofresidues 549-838 (FIG. 1D). FGFR3 is a member of the FGFR receptortyrosine kinase (TK) family that transduces intracellular signals afterbinding to FGF ligands (Turner and Grose, 2010). TACC3 belongs to theevolutionarily conserved TACC gene family, which also includes TACC1 andTACC2. The distinctive feature of TACC proteins is the presence of acoiled-coil domain at the C-terminus, known as the TACC domain. Throughthe TACC domain, TACC proteins localize to the mitotic spindle duringmetaphase and stabilize the microtubule spindle network (Hood and Royle,2011; Peset and Vernos, 2008). In the predicted fusion protein theintracellular TK domain of FGFR3 is fused upstream of the TACC domain ofTACC3 (FIG. 1D).

Exon-specific gene expression analysis from the RNA-seq coverage inGSC-1123 demonstrated that the FGFR3 and TACC3 exons implicated in thefusion are highly overexpressed compared with the mRNA sequences notincluded in the fusion event (FIG. 10A). Quantitative RT-PCR showed thatthe expression of the fused FGFR3-TACC3 exons is significantly higher inGSC-1123 than other GSCs and the normal brain (80 to 130-fold, FIG.10B). Without being bound by theory, functionally significant geneticrearrangements may result in marked overexpression (outlier) of thegenes implicated in the fusion events (Tomlins et al., 2007; Tomlins etal., 2005). The FGFR3-TACC3 fusion protein was also abundantly expressedin GSC-1123 and in the primary tumor GBM-1123, as shown by Western blotand immunohistochemistry (FIGS. 10C and 10D). On a Western Blot, theFGFR3-TACC3 fusion protein migrated at a size of ˜150 kD andimmunoprecipitation followed by mass spectrometry revealed the presenceof FGFR3 and TACC3 peptides consistent with the cDNA translationprediction (FIG. 10E). Using PCR, the genomic breakpoint coordinateswere mapped to chromosome 4 (#1,808,966 for FGFR3 and #1,737,080 forTACC3, genome build GRCh37/hg19) falling within FGFR3 exon 17 and TACC3intron 7, which gives rise to a transcript in which the 5′ FGFR3 exon 16is spliced to the 3′ TACC3 exon 8. The DNA junctions of FGFR3 and TACC3show microhomology within a 10-base region, an observation consistentwith results previously reported for other chromosomal rearrangements inhuman cancer (Bass et al., 2011; Stephens et al., 2009) (FIG. 1E).

The experimental validation of the inferred genomic fusions was focusedon FGFR3-TACC3. Exome-Fuse identified FGFR3-TACC3 gene fusions in fourGBM samples with breakpoints spanning invariably within intron 16 ofFGFR3 (which is downstream to the coding region for the TK domain) andintron 7-10 of TACC3 (which is upstream to the TACC domain) (FIG. 2A,Tables 4 and 5). Among the four positive TCGA GBM specimens, two wereavailable from TCGA centers for molecular analysis (TCGA-27-1835 andTCGA-06-6390) and, by Sanger sequencing, each of them were confirmed tocarry an in-frame fusion transcript that is consistent with thepredicted genomic breakpoints (FIGS. 2B and 2C). Thus, the frames of theFGFR3-TACC3 fusion proteins invariably result in juxtaposing the TKdomain of FGFR3 upstream of the TACC domain of TACC3. Consistent withthe abundant expression of FGFR3-TACC3 in GSC-1123 and GBM-1123, themRNA expression analysis of the TCGA tumors revealed that the fourFGFR3-TACC3-positive GBM display marked co-outlier expression of FGFR3and TACC3 (FIG. 2D). Recurrent gene fusions can be associated with localcopy number variations (CNV) of the breakpoint regions (Wang et al.,2009). Accordingly, the analysis of SNP arrays in the TCGA datasetrevealed the presence of microamplification events of the FGFR3 andTACC3 genes in all four FGFR3-TACC3-positive GBM (FIG. 2E).

TABLE 4 List of split inserts supporting the identificationof FGFR3-TACC3 fusion genes in four GBM samplesfrom the ATLAS-TCGA exome collection(SEQ ID NOS 187-224, respectively, in order of appearance) gene1 readread hg18 hg18 bit TCGAsampleID gene1 length readID %identity lengthmismatch gap start end genome genome e-value score read2 fastaTCGA-06-6390 FGFR3 76 C01PRACXX110828:1:1301:1934:116558 100 76 0 0 1 761778372 1778447 8E-40 151 GTGCTGGCATGCCGCGCC CTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGA CCTGGACCGTGTCCTTAC CGTG TCGA-06-6390 FGFR3 76C01RDACXX110828:3:2305:4872:47002 98.68 76 1 0 1 76 1778364 17784392E-37 143 ATGCGGGAGTGCTGGCAT GACGCGCCCTCCCAGAGG CCCACCTTCAAGCAGCTGGTGGAGGACCTGGACCGT GTCC TCGA-06-6390 FGFR3 76D03U9ACXX110625:6:1203:16178:138219 100 76 0 0 1 76 1778413 17784888E-40 151 AGCTGGTGGAGGACCTGG ACCGTGTCCTTACCGTGA CGTCCACCGACGTGAGTGCTGGCTCTGGCCTGGTGC CACC TCGA-06-6390 TACC3 76C01PRACXX110829:2:1102:13552:120312 100 76 0 0 1 76 1708918 17088438E-40 151 CCCTTAAAACAACTCGTT CCCTCAGACCACACACAA GACAGTTCAAGAGGGACTCAAGGACTTACAGGAATG TCCA TCGA-06-6390 TACC3 76C01PRACXX11D628:8:2308:6515:60354 100 76 0 0 1 76 1709956 1708881 8E-40151 AACCAAAGGCTCAGACCC CCAGGAATAGAAAATATA GGCCCTTAAAACAACTCGTTCCCTCAGACCACACAC AAGA TCGA-06-6390 TACC3 76C01RDACXX110628:6:1305:16843:57213 98.68 76 1 0 1 76 1708865 17087802E-37 143 TCAAGGACTTACAGGAAT GTCCAGTGCTCCCAAGAA ATCGAACTCCACAAGCTTGGCTTCCCGCGCACGTCC TGAG TCGA-06-6390 TACC3 75D03U9ACXX110625:2:1202:19578:90281 100 75 0 0 1 75 1708861 1708787 3E-39149 GGACTTACAGGAATGTCC AGTGCTCCCAAGAAATCG AACTCCACAAGCTTGGCTTCCCGCGGACGTCCTGAG GGAT TCGA-06-6390 TACC3 76D03U9ACXX110625:4:2306:2024:174970 100 76 0 0 1 76 1708869 1708821 8E-40151 CAGACCACACACAAGACA GTTCAAGAGGGACTCAAG GACTTACAGGAATGTCCAGTGCTCCCAAGAAATCGA ACTC TCGA-12-0826 FGFR3 7261C59AAXX100217:4:21:17613:20556 98.61 72 0 1 1 71 1778439 1778510 2E-34133 CTTACCGTGACGTCCACC GACGTGAGTGCTGGCTCT GGCCTGGTGCCACCCGCCTATGCCCCTCCCCTGCCC TTAG TCGA-12-0826 TACC3 7542MJNAAXX090813:5:30:1412:128060 100 75 0 0 2 76 1707299 1707225 3E-39149 AAACTTGAGGTATAAGGA CTGCTTCCTCAAGGCCGA CTCCTTAAACTGGGGACAAGAGGGCAAGTGATCAGG TCTG TCGA-12-0826 TACC3 7661C59AAXX1002:17:4:2:4279:6948 100 76 0 0 1 76 1707209 1707224 8E-40 151AACTTGAGGTATAAGGAC TGCTTGGTGAAGGCCGAC TCCTTAAACTGGGGACAAGAGGGCAAGTGATCAGGT CTGA TCGA-12-0826 FGFR3 7642MJNAAXX090813:5:37:435:1250#0 100 76 0 0 1 76 1778340 1778421 8E-40151 GCCCGCAGGTACATGATC ATGCGGGAGTGCTGGCAT GCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTG GTGG TCGA-12-0826 FGFR3 5161C59AAXX100217:5:52:7727:2557 98.04 51 1 0 1 51 1778443 1778426 4E-2493.7 ACCGTGACGTCCACCGAC GTGAGTGCTGGCTCTGGC CTGGTGCGACCCGCCGATCTCTCTCCCCTGTCCTTT TCCT TCGA-19-5958 TACC3 62D03U9ACXX110825:4:2208:9451:114169 90.32 62 6 0 1 62 1707141 17072024E-17 75.6 TGGGAGGGTGCGGGGGGC CGGGGGGGGGAGTGTGCA GGTGAGCTCCCTGGCCCTTGGCCCCCTGCCCTCTGG GGGG TCGA-19-5958 TACC3 74D03U9ACXX110825:1:2204:20084:21192 98.95 74 3 0 1 74 1707097 17071705E-33 123 CTGGGAATGGTGGTGTCT CGGGCAGGGTTGTGGGTG ACCGGGGGTGGGAGGGTGCGGGGGACCGGGGGGGGG AGGG TCGA-27-1835 FGFR3 76C00HWAEXX110325:7:2202:17660:110656 100 76 0 0 1 76 1778338 17784138E-40 151 AGCGCCCTGCCCGCAGGT ACATGATCATGCGGGAGT GCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCA AGCA TCGA-27-1835 TACC3 76C00HWAEXX110325:7:1104:10731:5183 100 76 0 0 1 76 1709417 1709417 8E-40151 GCCAACGCCATGCCCAGG CCGGAGAGTCCCGGGGAG GCTGCTGGTGGGCAGCTGACTGCGGGGACACTGGGT GGAA TCGA-27-1835 TACC3 60B0972ABXX11D408:2:2201:5911:24541 100 60 0 0 1 60 1709445 1709445 3E-30119 AGGCCACCAGAGGCCAAC GCCATGCCCAGGCCGGAG AGTCCCGGGGAGGCTGCTGGTGGGGAGGCGAACGCG GGGA TCGA-27-1835 TACC3 61B097UABXX110405:4:2102:15742:63594 91.8 61 5 0 1 61 1709422 17094226E-19 91.9 TGCCCAGGCCGGAGAGTC CCGGGGCGGCTGCTGGGG GGGAGCTGACTGGGGGGGCACTGGGGGGGAGACCCG GGCC hg18 hg18 gene2 read read genome genome bitTCGAsampleID gene2 length readID %identity length mismatch gap start endstart end e-value score read2 fasta TCGA-06-6390 TACC3 76C01FRACXX110628:1:1301:1934:116558 100 76 0 0 1 76 1708922 1708847 8E-40151 TAGGCCCTTAAAACAACT CGTTCCCTCAGACCACAC ACAAGACAGTTCAAGAGGGACTCAAGGACTTACAGG AATG TCGA-06-6390 TACC3 76C01RDACXX110629:3:2305:4872:47008 100 76 0 0 1 76 1708867 1708792 8E-40151 ACTCAAGGACTTACAGGA ATGTCCAGTGCTCCCAAG AAATCGAACTCCACAAGCTTGGCTTCCCGCGGACGT CCTG TCGA-06-6390 TACC3 76D03U9ACXX110625:6:1203:16178:138219 100 76 0 0 1 76 1708921 17088468E-40 151 AGGCCCTTAAAACAACTC GTTCCCTCAGACCACACA CAAGACAGTTCAAGAGGGACTCAAGGACTTACAGGA ATGT TCGA-06-6390 FGFR3 76C01PRACXX110828:2:1102:13552:120312 100 76 0 0 1 76 1778387 17784628E-40 151 GCCCTCCCAGAGGCCCAC CTTCAAGCAGCTGGTGGA GGACCTGGACCGTGTCCTTACCGTGACGTCCACCGA CGTG TCGA-06-6390 FGFR3 76C01PRACXX110528:8:2308:6515:60354 100 76 0 0 1 76 1778382 1778457 8E-40151 GCCGCGCCCTCCCAGAGG CCCACCTTCAAGCAGCTG GTGGAGGACCTGGACCGTGTCCTTACCGTGACGTCC ACCG TCGA-06-6390 FGFR3 76C01RDACXX110628:6:1305:16843:57213 96.05 76 3 0 1 76 1778417 17784921E-32 127 GGTGGAGGACCTGGACCG TGACCTTACCGGGACGTC CACCGACGGGAGTGCTGGCTCTGGCCTGGTGCCACC CGCC TCGA-06-6390 FGFR3 76D03U9ACXX110525:2:1202:19578:90281 100 76 0 0 1 76 1778447 1776522 8E-40151 GACGTCCACCGACGTGAG TGCTGGCTCTGGCCTGGT GCCACCCGCCTATGCCCCTCCCCCTGCCGTCCCCGG CCAT TCGA-06-6390 FGFR3 76D03U9ACXX110525:4:2306:2694:174970 100 76 0 0 1 76 1778435 1778510 8E-40151 TGTCCTTACCGTGACGTC CACCGACGTGAGTGCTGG CTCTGGCCTGGTGCCACCCGCCTATGCCCCTCCCCC TGCC TCGA-12-0826 TACC3 7261C59AAXX100217:4:21:17613:20886 95.83 72 3 0 1 72 1707352 1707221 3E-30119 TACCTGCTGGTCTCGGTG GCCACGGGCACTGGTCTA CCAGGGCTGTCCCTCCGGAGGGGGTCAAACTTGAGG GATA TCGA-12-0826 FGFR3 7642MJNAAXX090813:5:30:1412:1280#0 98.68 76 1 0 1 76 1778427 1778502 2E-37143 CTGGACCGTGTCCTTACC GTGACGTCCACCGACGTG AGTGCTGGCTCTGGCCTGGTGCCACCCGCCCATGCC CCTC TCGA-12-0826 FGFR3 7661C59AAXX100217:4:2:4279:6949 98.68 75 0 1 1 75 1778435 1776510 8E-37141 TGTCCTTACCGTGACGTC CACCGACGTGAGTGCTGG CTCTGGCCTGGTGCCACCCGCCTATGCCCCTCCCCT GCCC TCGA-12-0826 TACC3 6742MJNAAXX090813:5:37:435:1250#0 98.51 67 1 0 1 67 1707635 1707569 5E-32125 AAAAGATTTAAGTTTAGA TCTTTAATATACCTAGAA CGGTGGCTGTAACCAGCAAGGCAGGAGCCCTTTGTG TTGG TCGA-12-0826 TACC3 7561C59AAXX100217:5:69:7727:2557 97.33 76 2 0 2 76 1707306 1707232 5E-36133 TGGGTCAAACTTGAGGTA TAAGGACTGCTTCCTCAA GGCCGACTCCTTATACTGGGGACAAGAGGGCAAGTG ATCA TCGA-19-5958 FGFR3 76D03U9ACXX110625:4:2206:9451:114168 98.68 76 1 0 1 76 1778462 17765372E-37 143 GAGTGCTGGCTCTGGCCT GGTGCCACCCGCCTATGC CCCTCCCCCTGGCGTCCCCGGCCATCCTGCCCCCCA GAGT TCGA-19-5958 FGFR3 76D03U9ACXX110625:1:2204:20064:21192 100 76 0 0 1 76 1778462 1776537 2E-41151 GAGTGCTGGCTCTGGCCT GGTGCCACCCGCCTATGC CCCTCCCCCTGCCGTCCCCGGCCATCCTGCCCCCCA GAGT TCGA-27-1835 TACC3 76C00HWABXX110325:7:2202:17680:110566 96.05 76 3 0 1 76 1709492 17094171E-32 127 GCCAACGCCATGCCCAGG CCGGAGAGTCCCGGGGAG GCTGCTGGTGGGGAGCTGACTTCGGGGACACTGGGG GGAA TCGA-27-1835 FGFR3 76C00HWABXX110325:7:1104:10731:5183 96.05 76 3 0 1 76 1778363 17764381E-32 127 CATGCGGGAGTGCTGGCA TGGCGCGCCCTCCCAGCG GCCCACCTTCAAGCAGCTGGTGGGGGACCTGGACCG TGTC TCGA-27-1835 FGFR3 76B09V2ABXX110408:2:2291:5811:24541 100 76 0 0 1 76 1778458 1778533 8E-40151 ACGTGAGTGCTGGCTCTG GCCTGGTGCCACCCGCCT ATGCCCCTCCCCCTGCCGTCCCCGGCCATCCTGCCC CCCA TCGA-27-1835 FGFR3 76B097UABXX110405:4:2102:15742:63594 100 76 0 0 1 76 1778388 1776453 8E-40151 CCCTCCCAGAGGCCCACC TTCAAGCAGCTGGTGGAG GACCTGGACCGTGTCCTTACCGTGACGTCCACCGAC GTGA

TABLE 5 List of split reads supporting the identification of FGFR3-TACC3fusion genes in four GBM samples from the ATLAS-TCGA exome collection(SEQ ID NOS 225-318, respectively, in order of appearance) samplegenesplit1 readID directionsplit hg18startsplit1 hg18stopsplit1 length1mismatch1 gap1 seqsplit TCGA-06-6390 TACC3D03U9ACXX110025:2:1202:19578:90281 R 1778521 1778521 1 0 0GGACTTACAGGAATGTCCAG TGCTCCCAAGAAATCGAACT CCACAAGCTTGGCTTCCCGCGGACGTCCTGAGGGA***T TCGA-06-6390 FGFR3 C01PRACXX11025:3:1104:10052:66371F 1778520 1778521 2 0 0 CA***TCCCTCAGGACGTCC GCGGGAAGCCAAGCTTGTGGAGTTCGATTTCTTGGGAGCA CTGGACATTCCTGTAAGTC TCGA-06-6390 FGFR3C01PRACXX11028:5:1108:3119:22892 F 1778520 1778521 2 0 0CA***TCCCTCAGGACGTCC GCGGGAAGCCAAGCTTGTGG AGTTCGATTTCTTGGGAGCACTGGACATTCCTGTAAGTC TCGA-06-6390 FGFR3D03I9ACXX110025:8:2304:13007:108832 F 1778520 1778521 2 0 0CA***TCCCTCAGGACGTCC GCGGGAAGCCAAGCTTGTGG AGTTCGATTTCTTGGGAGCACTGGACATTCCTGTAAGTC TCGA-06-6390 FGFR3 C01PRACXX11028:5:2108:1999:91559F 1778518 1778521 4 0 0 GCCA***TCCCTCAGGACGT CCGCGGGAAGCCAAGCTTGTGGAGTTCGATTTCTTGGGAG CACTGGACATTCCTGTAAG TCGA-06-6390 FGFR3C01RDACXX110529:3:1336:1446:68211 F 1778515 1778521 7 0 0CCGGCCA***TCCCTGAGGA CGTCCGCGGGAAGCCAAGCT TGTGGAGTTCGATTTCTTGGGAGCACTGGACATTCCTGT TCGA-06-6390 TACC3D03U9ACXX110605:5:2205:12523:196352 R 1778514 1778521 8 1 0CAGGAATGTCCAGTGCTACC AAGAAATCGAACTCCACAAG CTTGGGTTCCCGCGGACGTCCTCCGGGA***TGGCCGTG TCGA-06-6390 TACC3 C01PRACXX110628:5:2103:6315:17943R 1778514 1778521 6 0 0 CAGGAATGTCCAGTGCTCCC AAGAAATCGAACTCCACAAGCTTGGCTTCCCGCGGACGTC CTGAGGGA***TGGCCGGG TCGA-06-6390 FGFR3C01PRACXX110629:3:1204:10831:2928 F 1778512 1778521 10 0 0TCCCCGGCCA***TCCCTCA GGACGTCCGCGGGAAGCCAA GCTTGTGGAGTTCGATTTCTTGGGAGCACTGGACATTCC TCGA-06-6390 FGFR3C01PRACXX110828:5:2209:6732:191360 F 1778512 1778521 10 0 0TCCCCGGCCA***TCCCTCA GGAAGTCCGCGGGAAGCCAA GCTTGTGGAGTTCGATTTCTTGGGAGCACTGGACATTCC TCGA-06-6390 FGFR3 C01PRACXX110628:8:1308:2911:20590F 1778511 1778521 11 0 0 GTCCCCGGCCA***TCCCTC AGGACGTCCGCGGGAAGCCAAGCTTGTGGAGTTCGATTTC TTGGGAGCACTGGACATTC TCGA-06-6390 FGFR3C01PRACXX110628:8:2207:4588:64017 F 1778509 1778521 13 0 0CCGTCCCCGGCCA***TCCC TCAGGACGTCCGCGGGAAGC CAAGCTTGTGGAGTTCGATTTCTTGGGAGCACTGGACAT TCGA-06-6390 TACC3 C01PRACXX110628::2205:11925:39734R 1778501 1778521 21 0 0 TGCTCCCAAGAAATCGAACT CCACAAGCTTGGCTTCCCGCGGACGTCCTGAGGGA***TG GCCGGGGACGGCAGGGGGA TCGA-06-6390 TACC3C01PRACXX110528:6:1105:12159:179489 R 1778494 1778521 28 0 0AAGAAATCGAACTCCACAAG CTTGGCTTCCCGCGGACGTC CTGAGGGA***TGGCCGGGGACGGCAGGGGGAGGGGCAT TCGA-06-6390 TACC3D03U9ACXX110625:4:2209:12501:40382 R 1778491 1778521 31 1 0AAATCGAACTCCACAAGCTT GGCTTCCCGCGGACGTCCTG AGGGA***TGGCCGGGGGCGGCAGGGGGAGGGGCATAGG TCGA-06-6390 FGFR3 C01PRACXX110628:3:1305:3044:13239F 1778473 1778521 49 0 0 CTGGCCTGGTGCCACCCGCC TATGCCCCTCCCCCTGCCGTCCCCGGCCA***TCCCTCAG GACGTCCGCGGGAAGCCAA TCGA-06-6390 TACC3D03U9ACXX110625:5:2205:12523:195352 R 1778470 1778521 52 4 0TCGTCCCGCGGACTTCCTGA TGGA***TCGCCGGGGACGG CAGGGGGAGGGGCATAGGCGTGTGGCACCAGGCCAGCTC TCGA-06-6390 TACC3C01PRACXX110528:7:2205:11825:39734 R 1778469 1778521 53 1 0CTTCCCGCGGACGTCCTGAG GGA***TGGCCGGGGACGGA AGGGGGAGGGGCATAGGCGGGTGGCACCAGGCCAGAGCC TCGA-06-6390 FGFR3D03U9ACXX110525:7:2100:4492:193350 F 1778464 1778521 58 0 0GTGCTGGCTCTGGCCTGGTG CCACCCGCCTATGCCCCTCC CCCTGCCGTCCCCGGCCA***TCCCTCAGGACGTCCGCG TCGA-06-6390 TACC3 C01PRACXX110628:5:2103:6815:17943R 1778452 1778521 70 0 0 GAGGGA***TGGCCGGGGAC GGCAGGGGGAGGGGCATAGGCGGGTGGCACCAGGCCAGAG CCAGCACTCACGTCGGTGG TCGA-12-0826 TACC3B1C5RAAXX1D0217:4:93:15133:6133 R 1778495 1778502 6 0 0GGACAAGAGGGCAAGTGATC AGGTCTGACTGCCATCCCCT AACACACACAGGGGGGCTAAGGGCAGGG***GAGGGGCA TCGA-12-0826 TACC3 B1C59AAXX100217:5:107:10875:15040R 1778495 1778502 8 0 0 GGACAAGAGGGCAAGTGATC AGGTCTGACTGCCATCCCCTAACACACACAGGGGGGCTAA GGGCAGGG***GAGGGGCA TCGA-12-0826 FGFR3B1C59AAXX100217:5:108:1909:11295 F 1778494 1778502 9 0 0ATGCCCCTC***CCCTGCCC TTAGCCCCCCTGTGTGTGTT AGGGGATGGCAGTCAGACCTGATCACTTGCCCTCTTGTC TCGA-12-0826 FGFR3 B1C59AAXX100217:5:82:13129:10637F 1778490 1778502 13 0 0 GCCTATGCCCCTC***CCCT GCCCTTAGCCCCCCTGTGTGTGTTAGGGGATGGCAGTCAG ACCTGATCACTTGCCCTCT TCGA-12-0826 FGFR342MJNAAXX090813:5:80:891:1877#0 F 1778481 1778502 22 0 0GTGCCACCCGCCTATGCCCC TC***CCCTGCCCTTAGCCC CCCTGTGTGTGTTAGGGGATGGCAGTCAGACCTGATCAC TCGA-12-0826 TACC3 B1C59AAXX100217:3:75:30598:12001R 1778470 1778502 33 1 0 TGACTGCCATCCCCTAACAC ACACAGGGGGGCTAAGGGCAGGG***GAGGGGCATAGGCG GGGGGCACCAGGCCAGAGC TCGA-12-0826 TACC3B1C59AAXX100217:4:114:5844:3101 R 1778470 1778502 33 1 0TGACTGCCATCCCCTAACAC ACACAGGGGGGCTAAGGGCA GGG***GAGGGGCATAGGCGGGGGGCACCAGGCCAGAGC TCGA-12-0826 TACC3 42MJNAAXX0908136:70:652:108#0 R1778458 1778502 37 3 0 TGCCATCCCCTAACACACAC AGGGGGGCTAAGGGCAGGG***GAGGGGCATAGGCGGGGG GCACCAGGACAGAGGCAGC TCGA-12-0826 TACC3B1C59AAXX100217:3:55:4955:15075 R 1778451 1778502 52 5 0CACACAGGGGGGCTAAGGGC AGGG***GAGGGGCATAGGC GGGGGGGACCAGGCCCGAGCCAGCACTCACGTCGGGGGG TCGA-12-0826 FGFR3 42MJNAAXX090813:5:23:158:1180#0 F1778447 1778502 56 0 0 GACGTCCACCGACGTGAGTG CTGGCTCTGGCCTGGTGCCACCCGCCTATGCCCCTCC*** CCCTGCCCTTAGACCCCCTG TCGA-12-0826 FGFR3B1C59AAXX100217:4:21:17013:20886 F 1778439 1778502 64 0 0CTTACCGTGACGTCCACCGA CGTGAGTGCTGGCTCTGGCC TGGTGCCACCCGCCTATGCCCCTC***CCCTGCCCTTAG TCGA-12-0826 FGFR3 B1C59AAXX100217:4:2:4279:5949 F1778438 1778502 68 0 0 TGTCCTTACCGTGACGTCCA CCGACGTGAGTGCTGGCTCTGGCCTGGTGCCACCCGCCTA TGCCCCTC***CCCTGCCC TCGA-19-5958 TACC3C01RDACXX110528:6:1102:11157:101952 R 1778533 1778539 7 0 0CGGGGGTGGGAGTGTGCGGG TGACCGGGGGTGGGAGTGTG CAGGTGACCTCCCTGGCCCTTAGCCCCCT***GCACTCT TCGA-19-5958 TACC3 C01REACXX110629:2:2104:6009:99392R 1778517 1778539 23 0 0 CGGGTGACCGGGGGAGGGAG TGTGCAGGGGACCTCCCTGAGGGTTAGCCCCCT***GCAC TCTGGGGGGCAGGATGGCC TCGA-19-5958 TACC3C01PRACXX110022:7:2103:12434:91988 R 1778501 1778539 39 0 0GGAGTGTGCAGGTGACCTCC CTGGCCCTTAGCCCCCT*** GCACTCTGGGGGGCAGGATGGCCGGGGACGGCAGGGGGA TCGA-27-1835 TACC3 B05UCABXX110322:5:1103:9252:48754R 1778558 1778598 10 1 0 GGGGAGGCTGCTGGTGGGCA GCTGACTGCGGGGACACTGGGAGGAAGCCTGGACCCTCAG CGAACT***TCGCCCAGCC TCGA-27-1835 FGFR3C00HWABXX110325:4:1201:20830:90877 F 1778557 1778598 20 0 0ACAGCCTGGGCACAGAGGTG GCTGTGCGA***AGGTCGCT GAGGGTCCAGGCTTCCACCCAGTGTCCCCGCAGTCAGCT TCGA-27-1835 TACC3B05UCABXX110322:5:1108:14043:83287 R 1778554 1778598 32 0 0TGACTGCGGGGACACTGGGT GGAAGCCTGGACCCTCAGCG ACCT***TCGCACAGCCACCTCTGTGCCCAGGCTGTGCC TCGA-27-1835 TACC3B097UABXX110405:4:2204:19443:99453 R 1778558 1778598 38 0 0CGGGGACACTGGGTGGAAGC CTGGACCCTCAGCGACCT** *TCGCACAGCCACCTCTGTGCCCAGGCTGTGCCCCAGAA TCGA-27-1835 TACC3B097UABXX110405:4:2201:20648:44401 R 1778557 1778598 39 2 0GGGGACACTGGGTGGAAGCC TGGACCCTCAGCGACCT*** TCGCACAGCCACCTCTGTGGCCAGGCTGTGCCACAGAAG TCGA-27-1835 TACC3B097UABXX110405:2:2104:15688:71022 R 1778555 1778598 41 0 0GGACACTGGGTGGAAGCCTG GACCCTCAGCGACCT***TC GCACAGCCACCTCTGTGCCCAGGCTGTGCCCCAGAAGGC TCGA-27-1835 TACC3C00HWABXX110325:5:2102:20394:42427 R 1778543 1778598 53 3 0GAAGCCTGGACCCTCAGCGA CCT***TCGCACAGCCACCT CTGTGCCCCGGCTGTGCCCCAGCCGGCCCGCCCCACACC TCGA-27-1835 TACC3B09V2ABXX110408:6:1203:19187:141962 R 1778543 1778598 53 0 0GAAGCCTGGACCCTCAGCGA CCT***TCGCACAGCCACCT CTGTGCCCAGGCTGTGCCCCAGAAGGCCCGCCCCACACC TCGA-27-1835 TACC3 B09V2ABXX110408:8:1205:4774:91604R 1778537 1778598 59 0 0 TGGACCCTCAGCGACCT*** TCGCACAGCCACCTCTGTGCCCAGGCTGTGCCCCAGAAGG CCCGCCCCACACCTCAGCA TCGA-27-1835 TACC3C00HWABXX110325:2:1107:16165:23614 R 1778535 1778598 61 0 0GACCCTCAGCGACCT***TC GCACAGCCACCTCTGTGCCC AGGCTGTGCCCCAGAAGGCCCGCCCCACACCTCAGCACT TCGA-27-1835 TACC3C00HWABXX110325:7:2107:1225:187363 R 1778530 1778598 66 0 0TCAGCGACCT***TCGCACA GCCACCTCTGTGCCCAGGCT GTGCCCCAGAAGGCCCGCCCCACACCTCAGCACTCTGGG TCGA-27-1835 TACC3 B097UABXX110405:2:214:15656:71022R 1778523 1778598 73 0 0 CCT***TCGCACAGCCACCT CTGTGCCCAGGCTGTGCCCCAGAAGGCCCGCCCCACACCT CAGCACTCTGGGGGGCAGG sample genesplit2 readIDdirectionsplit hg18startsplit2 hg18stopsplit2 length2 mismatch2 gap2seqmate TCGA-06-6390 FGFR3 D03U9ACXX110025:2:1202:19578:90281 R 17087871708851 75 0 0 GACGTCCACCGACGTGAGTG CTGGCTCTGGCCTGGTGCCACCCGCCTATGCCCCTCCCCC TGCCGTCCCCGGCCAT TCGA-06-6390 TACC3C01PRACXX11025:3:1104:10052:66371 F 1708787 1708850 74 0 0CAAGAGGGACTCAAGGACTT ACAGGAATGTCCAGTGCTCC CAAGAAATCGAACTCCACAAGCTTGGCTTCCCGCGG TCGA-06-6390 TACC3 C01PRACXX11028:5:1108:3119:22892 F1708787 1708850 74 0 0 CAAGAGGGACTCAAGGACTT ACAGGAATGTCCAGTGCTCCCAAGAAATCGAACTCCACAA GCTTGGCTTCCCGCGG TCGA-06-6390 TACC3D03I9ACXX110025:8:2304:13007:108832 F 1708787 1708850 74 0 0ATAGGCCCTTAAAACAACTC GTTCCCTCAGACCACACACA AGACAGTTCAAGAGGGACTCAAGGACTTACAGGAAT TCGA-06-6390 TACC3 C01PRACXX11028:5:2108:1999:91559 F1708787 1708858 72 0 0 TCAAGAGGGACTCAAGGACT TACAGGAATGTCCAGTGCTCCCAAGAAATCGAACTCCACA AGCTTGGCTTCCCGCG TCGA-06-6390 TACC3C01RDACXX110529:3:1336:1446:68211 F 1708787 1705855 69 0 0ACCACACACAAGACAGTTCA AGAGGGACTCAAGGACTTAC AGGAATGTCCAGTGCTCCCAAGAAATCGAACTCCAC TCGA-06-6390 FGFR3 D03U9ACXX110605:5:2205:12523:196352R 1708787 1705854 68 4 0 GAGCTGGCCTGGTGCCACAC GCCTATGCCCCTCCCCCTGCCGTCCCCGGCGATCCATCAG GAAGTCCGCGGGACGA TCGA-06-6390 FGFR3C01PRACXX110628:5:2103:6315:17943 R 1708787 1705854 68 0 0CCACCGACGTGAGTGCTGGC TCTGGCCTGGTGCCACCCGC CTATGCCCCTCCCCCTGCCGTCCCCGGCCATCCCTC TCGA-06-6390 TACC3 C01PRACXX110629:3:1204:10831:2928 F1708787 1703852 66 0 0 CAAGAGCCTCAGACAGTGCA TGAGGGACCCGAGACAGTGCGGCGAGGGAACAGCACAGCG GCCCCATGCCCCCAAC TCGA-06-6390 TACC3C01PRACXX110828:5:2209:6732:191360 F 1708787 1703852 66 1 0CAAGAGCCTCAGACAGTGCA TGAGGGACCCGAGACAGTGC GGCGAGGGAACAGCACAGGGGCCCCATGCCCCCAAC TCGA-06-6390 TACC3 C01PRACXX110628:8:1308:2911:20590 F1708787 1708851 65 0 0 CGTTCCCTCAGACCACACAC AAGACAGTTCAAGAGGGACTCAAGGACTTACAGGAATGTC CAGTGCTCCCAAGAGA TCGA-06-6390 TACC3C01PRACXX110628:8:2207:4588:64017 F 1708787 1708840 63 0 0CCAGGAATAGAAAATATAGG CCCTTAAAACAACTCGTTCC CTCAGACCACACACAAGACAGTTCAAGAGGGACTCA TCGA-06-6390 FGFR3 C01PRACXX110628::2205:11925:39734 R1708787 1708841 55 0 0 GGCTCTGGCCTGGTGCCACC CGCCTATGCCCCTCCCCCTTCCGTCCCCGGCCATCCCTCA GGACGTCCGCGGGAAG TCGA-06-6390 FGFR3C01PRACXX110528:6:1105:12159:179489 R 1708787 1708834 48 0 0GCCCTGCCCGCAGGTACATG ATCATGCGGGAGTGCTGGCA TGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCT TCGA-06-6390 FGFR3 D03U9ACXX110625:4:2209:12501:40382 R1708787 1708831 45 0 0 CTGGCATGCCGCGCCCTCCC AGAGGCCCACCTTTAAGCAGCTGGTAGAGGGCCTGGACCG TGTCCTTACCGTGACG TCGA-06-6390 TACC3C01PRACXX110628:3:1305:3044:13239 F 1708787 1708813 27 0 0TAAAACAACTCGTTCCCTCA GACCACACACAAGACAGTTC AAGAGGGACTCAAGGACTTACAGGAATGTCCAGTGC TCGA-06-6390 FGFR3 D03U9ACXX110625:5:2205:12523:195352R 1708787 1708810 24 4 0 CACGGCCATCCCGGAGGACG TCCGCGGGAACCCAAGCTTGTGGAGTTCGATTTCTTGGTA GCACTGGACATTCCTG TCGA-06-6390 FGFR3C01PRACXX110528:7:2205:11825:39734 R 1708787 1708809 23 0 0TCCCCCTGCCGTCCCCGGCC ATCCCTCAGGACGTCCGCGG GAAGCCAAGCTTGTGGAGTTCGATTTCTTGGGAGCA TCGA-06-6390 TACC3 D03U9ACXX110525:7:2100:4492:193350 F1708787 1705804 18 0 0 AGACCACACACAAGACAGTT CAAGAGGGACTCAAGGACTTACAGGAATGTCCAGTGCTCC CAAGAAATCGAACTCC TCGA-06-6390 FGFR3C01PRACXX110628:5:2103:6815:17943 R 1708787 1708702 6 0 0CCCGGCCATCCCTCAGGACG TCCGCGGGAAGCCAAGCTTG TGGAGTTCGATTTCTTGGGAGCACTGGACATTCCTG TCGA-12-0826 FGFR3 B1C5RAAXX1D0217:4:93:15133:6133 R1707185 1707253 69 2 1 GGCATGCCGCGCCCTCCCAG AGGCCCACCTTCAAGCAGCTGGTGGAGGACCTGGACCGTG TCCTTACCGTGACGTC TCGA-12-0826 FGFR3B1C59AAXX100217:5:107:10875:15040 R 1707185 1707253 69 2 1GGCATGCCGCGCCCTCCCAG AGGCCCACCTTCAAGCAGCT GGTGGAGGACCTGGACCGTGTCCTTACCGTGACGTC TCGA-12-0826 TACC3 B1C59AAXX100217:5:108:1909:11295 F1707185 1707252 68 2 1 CGGCGCACATACCTGCTGGT CTCGGTGGCCACGGGCACTGGTCTACCAGGACTGTCCCTC AGGAGGGGGTCAAACT TCGA-12-0826 TACC3B1C59AAXX100217:5:82:13129:10637 F 1707185 1707248 64 2 1ATACCTGCTGGTCTCGGTGG CCACGGGCACTGGTCTACCA GGACTGTCCCTCAGGAGGGGGTCAAACTTGAGGTAT TCGA-12-0826 TACC3 42MJNAAXX090813:5:80:891:1877#0 F1707185 1707239 55 2 1 AGGTATAAGGACTGCTTCCT CAAGGCCGACTCCTTAAACTGGGGACAAGAGGGCAAGTGA TCAGGTCTGACTGCCA TCGA-12-0826 FGFR3B1C59AAXX100217:3:75:30598:12001 R 1707185 1707228 44 2 1GGAGGACCTGGACTGTGTCC TTACCGTGACGTCCACCGAC GTGAGTGCTGGCTCTGGCCTGGTGCCACCCGCCTAT TCGA-12-0826 FGFR3 B1C59AAXX100217:4:114:5844:3101 R1707185 1707228 44 2 1 GGAGGACCTGGACCGTGTCC TTACCGTGACGTCCACCGACGTGAGTGCTGGCTCTGGCCT GGTGCCACCCGCCTAT TCGA-12-0826 FGFR342MJNAAXX0908136:70:652:108#0 R 1707185 1707224 40 2 1CAAGCAGCTGGTGGAGGACC TGGACCGTGTCCTTACCGTG ACGTCCACCGACGTGAGTGCTGGCTCTGGCCTGGTG TCGA-12-0826 FGFR3 B1C59AAXX100217:3:55:4955:15075 R1707185 1707209 25 2 1 ACCTTCAAGCAGCTGGTGGA GGACCTGGACCGTGTCCTTACCGTGACGTCCACCGACGTG AGTGCTGGCTCTGGCC TCGA-12-0826 TACC342MJNAAXX090813:5:23:158:1180#0 F 1707185 1707205 21 2 1CAAACTTGAGGTATAAGGAC TGCTTCCTCAAGGCCGACTC CTTAAACTGGGGACAAGAGGGCAAGTGATCAGGTCT TCGA-12-0826 TACC3 B1C59AAXX100217:4:21:17013:20886 F1707185 1707197 13 0 1 TACCTGCTGGTCTCGGTGGC CACGGGCACTGGTCTACCAGGGCTGTCCCTCCGGAGGGGG TCAAACTTGAGGGATA TCGA-12-0826 TACC3B1C59AAXX100217:4:2:4279:5949 F 1707185 1707193 8 0 1AACTTGAGGTATAAGGACTG CTTCCTCAAGGCCGACTCCT TAAACTGGGGACAAGAGGGCAAGTGATCAGGTCTGA TCGA-19-5958 FGFR3 C01RDACXX110528:6:1102:11157:101952R 1707202 1707270 69 1 0 AGCTGGTGGAGGACCTGGAC CGTGTCCTTACCGTGACGTCCACCGACGTGAGTGCTGGCT CTGGCCTGGTGCCACC TCGA-19-5958 FGFR3C01REACXX110629:2:2104:6009:99392 R 1707202 1707254 53 3 0GCGCCCTCCCAGAGGCCCAC CTTCAAGCAGCTGGTGGAGG ACCTGGACCGTGTCCTTACCGTGACGTCCACCGACG TCGA-19-5958 FGFR3 C01PRACXX110022:7:2103:12434:91988 R1707202 1707238 37 1 0 GCGGGAGTGCTGGCATGCCG CGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGA CCTGGACCGTGTCCTT TCGA-27-1835 FGFR3B05UCABXX110322:5:1103:9252:48754 R 1709397 1709452 66 2 0CCTCCACTGGGTCCTCAGGG GTGGGGGTCCCTCCGGGGCT GGGCGGGGGAGGGACTGGCAGGCCTGCAGGGGGGTT TCGA-27-1835 TACC3 C00HWABXX110325:4:1201:20830:90877 F1709397 1709443 47 0 0 TCACGGCAGCAAGAACCACA CTCACTGCTGCAAGGCCACCAGAGGCCAACGCCATGCCCA GGCCGGAGAGTCCCGG TCGA-27-1835 FGFR3B05UCABXX110322:5:1108:14043:83287 R 1709397 1709440 44 0 0TACATGATCATGCGGGAGGG CTGGCATGCCGCGCCCTCCC AGAGGCCCACCTTCAAGCAGCTGGTGGAGGGCCGGG TCGA-27-1835 FGFR3 B097UABXX110405:4:2204:19443:99453 R1709397 1709434 38 0 0 GGTGGGAAGCGGCGGGGCTC ACTCCTGAGCGCCCTGCCCGCAGGGACATGATCATGCGGG GGTGCTGGCCTTGCGG TCGA-27-1835 FGFR3B097UABXX110405:4:2201:20648:44401 R 1709397 1709433 37 0 0GCGCCCTCCCAGAGGCCCAC CTTCAAGCAGCTGGTGGAGG ACCTGGACCGTGTCCTTACCGTGACGTCCACCGACG TCGA-27-1835 FGFR3 B097UABXX110405:2:2104:15688:71022 R1709397 1709431 35 0 0 CCTGCCCCCCAGAGTGCTGA GGTGTGGGGCGGGCCTTCTGGGGCACAGCCTGGGCACAGA GGTGGCTGTGCGAAGG TCGA-27-1835 FGFR3C00HWABXX110325:5:2102:20394:42427 R 1709397 1709419 23 0 0GCAGGTACATGATCATGCGG GAGTGCCGGCATTTCGGGAC CTTCCCTCGGGCCACCCTCTTCCGGTTGTTGTGGGC TCGA-27-1835 FGFR3 B09V2ABXX110408:6:1203:19187:141962R 1709397 1709419 23 0 0 GCAGGTACATGATCATGCGG GAGTGCTGGCATGCCGCGCCCTCCCCGAGGACCACCTTCC AGCAGCCGGGGGAGGG TCGA-27-1835 FGFR3B09V2ABXX110408:8:1205:4774:91604 R 1709397 1709413 17 0 0CCCGAATAAGGTGGGAAGCG GCGGGGCTCACTCCTGAGCG CCCTGACCGCAGGTACATGAGCATGCGGGAGTGGCG TCGA-27-1835 FGFR3 C00HWABXX110325:2:1107:16165:23614 R1709397 1709411 16 0 0 CGTGTCCTTACCGTGACGTC CACCGACGTGAGTGCTGGCTCTGGCCTGGTGCCACCCGCC TATGCCCCTCCCCCTG TCGA-27-1835 FGFR3C00HWABXX110325:7:2107:1225:187363 R 1709397 1709400 10 0 0ACATGATCATGCGGGAGTGC TGGCATGCCGCGCCCCCCCA GAGGCCCACCTTCAAGCAGCTGGTGGAGGACCTGGA TCGA-27-1835 FGFR3 B097UABXX110405:2:214:15656:71022 R1709397 1709390 3 0 0 GCCTTCTGGGGCACAGCCTG GGCACAGAGGTGGCTGTGCGAAGGTCGCTGAGGGTCCAGG CTTCCACCCAGTGTCC

The FGFR3 and TACC3 genes are located 48-Kb apart on human chromosome4p16. The other members of the FGFR and TACC families retain the closephysical association of FGFR3 and TACC3, with FGFR1 and TACC1 paired onchromosome 8p11 and FGFR2 and TACC2 paired on chromosome 10q26. Withoutbeing bound by theory, the ancestral FGFR and TACC genes were physicallylinked and that this tandem gene cluster was duplicated at least twiceto generate the FGFR1-TACC1, FGFR2-TACC2 and FGFR3-TACC3 pairs that markmammalian evolution (Still et al., 1999). The highly conserved TKdomains among FGFR genes and TACC domains among TACC genes together withtheir invariable fusion in the FGFR3-TACC3 rearrangements prompted toask whether other intrachromosomal FGFR-TACC fusion combinations existin human GBM.

cDNA from a panel of 88 primary GBM were screened using pairs ofupstream PCR primers that bind the amino-terminal coding region of theTK domains of FGFR1, FGFR2 and FGFR3 and downstream primers that bind tothe carboxy-terminal coding region of the TACC domains of TACC1, TACC2and TACC3 genes, respectively. The screening resulted in theidentification of intrachromosomal FGFR-TACC fusions in two additionalcases (one harboring FGFR1-TACC1 and one FGFR3-TACC3), corresponding tothree of 97 total GBM (3.1%), including the GBM-1123 case. TheFGFR1-TACC1 fusion breakpoint in GBM-51 joined in-frame exon 17 of FGFR1to exon 7 of TACC1, resulting in a novel protein in which the TK domainof FGFR1 is fused upstream of the TACC domain of TACC1 (FIG. 2F). Thesame structure was conserved again in GBM-22 in which exon 16 of FGFR3is joined in-frame to exon 10 of TACC3 (FIG. 2G). None of the tumorsharboring FGFR-TACC fusions had mutations in IDH1 or IDH2 genes, thusindicating that FGFR-TACC-positive GBM mark an independent subgroup ofpatients from those carrying IDH mutations (Table 6) (Yan et al., 2009).The constant linkage of the FGFR-TK to the TACC domain created in eachof the seven GBM harboring FGFR-TACC rearrangements suggests thatFGFR-TACC fusion proteins may generate important functional consequencesfor oncogenesis in the brain.

TABLE 6 Age at initial IDH1-2 status IDH1-2 status Samples Type TimeStatus pathologic diagnosis (Sanger) (exome) TCGA-12-0826 FGFR3-TACC3845 DECEASED 38 WT WT TCGA-27-1635 FGFR3-TACC3 646 DECEASED 53 NA WTTCGA-19-5958 FGFR3-TACC3 164 LIVING 56 NA WT TCGA-06-6390 FGFR3-TACC3163 DECEASED 58 WT WT GBM-22 FGFR3-TACC3 390 DECEASED 60 WT NA GBM-1123FGFR3-TACC3 NA DECEASED 62 WT NA GBM-51 FGFR1-TACC1 NA NA NA WT NA Time= Survival (days after diagnosis) Sanger = analysis done by Sangersequencing of genomic DNA Exome = ainalysis done by the SAVI(Statistical Algorithm for Variant Identification), an algorithmdeveloped to detect point mutation in cancer (BRAF Mutations inHairy-Cell Leukemia, Tiacci E et al. The New England Journal of Medicine2011 Jun 16; 364(24): 2305-15) NA = Not Available WT = Wild typesequence for R132 and R172 of IDH1 and IDH2, respectively

Transforming Activity of FGFR-TACC Fusions.

To test the functional importance of the FGFR-TACC fusions in GBM, theFGFR3-TACC3 cDNA was cloned from GSC-1123 and recombinant lentiviruseswere prepared expressing FGFR3-TACC3, FGFR1-TACC1, a kinase-deadFGFR3-TACC3 protein (FGFR3-TACC3-K508M), wild type FGFR3 and wild typeTACC3. Transduction of Rat1A fibroblasts and Ink4A;Arf−/− astrocyteswith the FGFR3-TACC3 lentivirus resulted in the expression of the fusionprotein at levels comparable to those present in GSC-1123 (FIG. 11).Having reconstituted in non-transformed cells the endogenous level ofthe FGFR-TACC protein that accumulates in GBM cells, it was determinedwhether it was sufficient to initiate oncogenic transformation in vitroand in vivo. Rat1A cells expressing FGFR3-TACC3 and FGFR1-TACC1 but notthose expressing FGFR3-TACC3-K508M, FGFR3, TACC3 or the empty lentivirusacquired the ability to grow in anchorage-independent conditions in softagar (FIG. 3A). Transduction of the same lentiviruses in primaryInk4A;Arf−/− astrocytes followed by subcutaneous injection intoimmunodeficient mice revealed that only astrocytes expressingFGFR3-TACC3 and FGFR1-TACC1 formed tumors. The tumors emerged in 100% ofthe mice injected with astrocytes expressing the fusion proteins andwere glioma-like lesions with strong positivity for Ki67,phospho-histone H3, nestin, GFAP and Olig2 (FIG. 3B).

Next, it was determined whether the FGFR3-TACC3 fusion protein isoncogenic when transduced to a small number of cells directly into thebrain of immunocompetent animals. A recently described mouse gliomamodel was used in which brain tumors are initiated by lentiviraltransduction of oncogenes and inactivation of p53 in the mouse brain(Marumoto et al., 2009). To target adult NSCs, the adult mousehippocampus was stereotactically transduced with purified lentivirusexpressing the FGFR3-TACC3 protein and shRNA against p53(pTomo-FGFR3-TACC3-shp53). Seven of eight mice (87.5%) transduced withFGFR3-TACC3 succumbed from malignant brain tumors within 240 days (FIG.3C). None of the mice transduced with a lentivirus expressing the mostfrequent gain-of-function mutation in GBM (the constitutively activeEGFRvIII, pTomo-EGFRvIII-shp53) or the pTomo-shp53 control lentivirusdied or developed clinical signs of brain tumors (FIG. 3C). TheFGFR3-TACC3 tumors were high-grade glioma with strong propensity toinvade the normal brain and stained positive for the glioma stem cellmarkers nestin and Olig2 and the glial marker GFAP. They were alsohighly positive for Ki67 and phospho-histone H3, thus displaying rapidtumor growth (FIG. 3D). The expression of FGFR3-TACC3 in the xenograftand intracranial tumor models was comparable to the expression of theendogenous protein in the human GSCs and tumor (FIGS. 11D, 11E and 11F).

These data show that FGFR-TACC fusion proteins possess transformingactivity in two independent cellular models and this activity is not theresult of the overexpression of individual FGFR and TACC genes. Theyalso show that direct transduction of the FGFR3-TACC3 protein to theadult mouse brain leads to efficient development of malignant glioma.

The FGFR-TACC Fusions Interfere with Mitotic Progression and InduceChromosome Missegregation and Aneuploidy.

To elucidate the mechanism by which the FGFR-TACC fusion drivesoncogenesis, it was explored whether it activates downstream FGFRsignaling. FGFR3-TACC3 failed to hyperactivate the canonical signalingevents downstream of FGFR (pERK and pAKT) in the presence or absence ofthe ligands FGF-1, FGF-2 or FGF-8 (Wesche et al., 2011) (FIGS. 12A, 12Band 12C). However, FGFR3-TACC3 displayed constitutive phosphorylation ofits TK domain and the adaptor protein FRS2, both of which were abolishedby the specific inhibitor of FGFR-associated TK activity PD173074(Mohammadi et al., 1998) or the K508M mutation (FIG. 4A). Thus,FGFR3-TACC3 gains constitutive kinase activity that is essential foroncogenic transformation but the downstream signaling of this aberrantactivity is distinct from the canonical signaling events downstream toFGFR. By driving the localization of the fusion protein, the TACC domaincan create entirely novel TK-dependent functions. The TACC domain isessential for the localization of TACC proteins to the mitotic spindle(Hood and Royle, 2011; Peset and Vernos, 2008). Confocal imaging showedthat FGFR3-TACC3 designed an arc-shaped structure bending over andencasing the metaphase spindle poles, frequently displaying asymmetrytowards one of the two poles and relocated to the midbody as cellsprogressed into the late stages of mitosis (telophase and cytokinesis)(FIGS. 4B and 12D). Conversely, the localization of TACC3 was restrictedto spindle microtubules and did not relocalize to the midbody (FIG.12E). Wild type FGFR3 lacked discrete localization patterns in mitosis(FIG. 12F).

The mitotic localization of FGFR3-TACC3 indicates that it may impact thefidelity of mitosis and perturb the accurate delivery of the diploidchromosomal content to daughter cells, thus generating aneuploidy.Mitotic progression of individual cells was examined invector-transduced and FGFR3-TACC3 expressing cells co-expressing histoneH2B-GFP by time-lapse microscopy. The average time from nuclear envelopebreakdown to anaphase onset was increased in cells expressingFGFR3-TACC3 in comparison with control cells. The mitotic delay wasfurther exacerbated by difficulties in completing cytokinesis (FIGS. 4Cand 4D).

Next, it was determined whether the expressions of the FGFR-TACC fusionproteins induce defects of chromosomal segregation. Quantitativeanalyses of mitoses revealed that cells expressing FGFR3-TACC3 orFGFR1-TACC1 exhibit a three to five fold increase of chromosomalsegregation errors than control cells. The most frequent mitoticaberrations triggered by the fusion proteins were misaligned chromosomesduring metaphase, lagging chromosomes at anaphase and chromosome bridgesthat impaired cytokinesis and generated micronuclei in the daughtercells (FIGS. 4E, 4F and 13A). Aberrations at the metaphase-anaphasetransition frequently lead to the inability of mitotic cells to maintaina metaphase arrest after treatment with a spindle poison. Over 18% ofcells expressing FGFR3-TACC3 displayed prematurely separated sisterchromatids in contrast with less than 3% in control, FGFR3 orTACC3-expressing cells (FIGS. 13B and 13C). Accordingly, cellsexpressing the fusion protein were unable to efficiently arrest inmetaphase after nocodazole treatment (FIG. 13D).

The above findings indicate that expression of the FGFR3-TACC3 fusionprotein may spark aneuploidy. Karyotype analysis revealed thatFGFR3-TACC3 increased over 2.5 fold the percent of aneuploidy and led tothe accumulation of cells with broad distribution of chromosome countsin comparison with cells transduced with empty vector, FGFR3 or TACC3(FIG. 5A). Accordingly, GSC-1123 contained aneuploid modal number ofchromosomes (49) and manifested a broad distribution of chromosomecounts characterized by 60% of metaphase spreads that deviate from themode (Table 7)

TABLE 7 Chromosome analysis by SKY of 20 cells from the GSC-1123 cultureCell # Chr # +1 +2 +3 t(3:14) +4 (−4) del(4) +5 +6 +7 del(7) +8 −9 +9−10 +10 +11 1 97 2 2 2 2 1 2 2 4 2 2 2 2 51 1 1 3 49 1 1 4 50 1 1 5 49 11 6 86 2 2 2 2 2 3 1 1 2 7 95 2 2 2 2 1 2 4 2 2 2 8 98 2 3 2 1 2 1 2 4 31 2 9 86 2 1 1 1 1 1 1 6 2 3 1 1 10 44 1 1 1 1 1 1 1 11 49 1 1 1 12 49 11 13 98 2 2 2 2 2 2 4 2 2 2 14 49 1 1 15 48 1 1 16 51 1 1 17 49 1 1 1850 1 1 1 19 49 1 1 20 49 1 1 Cell # Chr # +12 +13 del(13) −14 +14 +15−16 +16 +17 +18 +19 +20 −21 +21 −22 +22 +X 1 97 2 2 2 2 2 2 2 3 4 4 2 22 2 51 2 2 1 1 1 3 49 1 1 1 1 4 50 1 1 1 1 1 5 49 1 1 1 1 6 86 2 2 2 2 22 4 3 3 1 2 7 95 2 2 2 2 2 2 2 4 2 4 2 2 2 8 98 2 2 2 2 2 2 2 3 4 4 3 22 9 86 2 2 2 2 1 4 3 1 2 1 2 10 44 1 1 1 1 1 11 49 1 1 1 12 49 1 1 1 113 98 2 2 2 2 2 2 4 4 4 2 2 2 14 49 1 1 1 1 15 48 1 1 1 1 16 51 1 1 1 21 17 49 1 1 1 1 18 50 1 1 1 1 1 19 49 1 1 1 20 49 1 1 1 1

Next, it was determined whether aneuploidy is a direct consequence ofFGFR3-TACC3 expression and is induced in human diploid neural cells.Primary human astrocytes analyzed six days after transduction with theFGFR3-TACC3 lentivirus exhibited a 5-fold increase of the rate ofaneuploidy and a significantly wider distribution of chromosome counts(FIGS. 5B, 5C and 5D). Consistent that aneuploidy is detrimental tocellular fitness, acute expression of FGFR3-TACC3 compromised theproliferation capacity of human astrocytes. However, continuous cultureof FGFR3-TACC3-expressing human astrocytes led to progressive gain ofproliferative capacity that overrode that of control cells (FIGS. 14A,14B). Thus, the acute expression of FGFR3-TACC3 in primary normal humancells from the central nervous system causes CIN and aneuploidy with anacute fitness cost manifested by slower proliferation.

It was also determined whether the CIN and aneuploidy caused byFGFR3-TACC3 requires the TK activity of FGFR3 and can be corrected.Treatment with PD173074 rescued the aneuploidy caused by FGFR3-TACC3 byover 80%, restored the narrow distribution of chromosome counts typicalof control cells and largely corrected the cohesion defect (FIGS. 6A, 6Band 6C). Together, these findings indicate that the CIN and aneuploidycaused by rearrangements of FGFR and TACC genes are reversible andsuggest that specific FGFR kinase inhibition may be a valuabletherapeutic strategy in tumor cells expressing FGFR-TACC fusionproteins.

FGFR-TACC Fusion Proteins are New Therapeutic Targets in GBM.

Driver genetic alterations trigger a state of oncogene addiction in thecancer cells harboring them that can be exploited therapeutically. Toask whether FGFR-TACC fusions confer addiction to FGFR-TK activity, cellgrowth was analyzed in the presence of PD173074, AZD4547 or BGJ398, thelatter being two highly specific inhibitors of FGFR-TK under clinicalinvestigation (Gavine et al., 2012; Guagnano et al., 2011). Each of thethree drugs inhibited growth of cells expressing FGFR3-TACC3 andFGFR1-TACC1 at concentrations<10 nM whereas they were ineffective atconcentrations as high as 1 μM in cells transduced with vector, FGFR3,TACC3 and the FGFR3-TACC3-K508M mutant (FIGS. 7A, 14C and 14D). Thesefindings underscore the elevated degree of specificity for FGFR kinaseinhibition towards cells carrying the fusion protein. The growth ofGSC-1123 cells, which naturally harbor the FGFR3-TACC3 translocation,was also abolished by nanomolar concentrations of FGFR-TK inhibitors(FIG. 7B). Targeting of the fusion gene by FGFR3 shRNA inhibited thegrowth of cells ectopically expressing FGFR3-TACC3 and GSC-1123proportionally to the silencing efficiency of FGFR3-TACC3 (FIGS. 7C and14E).

Finally, it was determined whether treatment with PD173074 of micebearing glioma xenografts of FGFR3-TACC3 transformed astrocytes inhibitstumor growth. Twelve days after injection of tumor cells, subcutaneoustumors were present in all animals. The mice were randomized in twocohorts and treated with PD173074 or vehicle. PD173074 elicited a potentgrowth inhibition of FGFR3-TACC3 glioma (FIG. 7D). To confirm theefficacy of a clinically meaningful FGFR-TK inhibitor using a moreanatomically relevant model, the AZD4547 FGFR inhibitor, a compoundunder clinical investigation (Gavine et al., 2012), was used againstintracranial luciferase-expressing FGFR3-TACC3-driven glioma xenografts.After an engraftment period, tumor-bearing animals were treated witheither AZD4547 or vehicle. Oral administration of AZD4547 markedlyprolonged survival (FIG. 7E). Taken together, the data provide a strongrationale for a clinical trial based on FGFR inhibitors in GBM harboringFGFR-TACC rearrangements.

Discussion

This work has established that recurrent, oncogenic and addicting genefusions identify a subset of GBM patients. The functionalcharacterization of FGFR-TACC fusions indicates that the constitutivelyactive FGFR-TK and the TACC domain of the fusion protein are bothessential for oncogenesis. The TACC-dependent mis-localization tomitotic cells of the FGFR kinase results in aberrantcompartmentalization of a constitutively active TK to the mitoticspindle pole, thus providing a mechanistic explanation for the impairedmitotic fidelity, chromosome mis-segregation and aneuploidy instigatedby the fusion protein.

Without being bound by theory, mutation of the genes that controlchromosome segregation during mitosis can explain the high rate of CINand aneuploidy, which is typical of most solid tumors including GBM(Gordon et al., 2012). A few examples of mutational inactivation ofcandidate genes have been reported in human cancer (Solomon et al.,2011; Thompson et al., 2010). However, gain-of-function mutationscausally implicated in the control of mitotic fidelity have not beendescribed. This clashes with the classical observation from cell fusionexperiments that the underlying mechanisms that cause CIN behave asdominant traits, indicating that the CIN phenotype results fromgain-of-function events rather than gene inactivation (Lengauer et al.,1997, 1998). The FGFR-TACC gene fusion is a novel mechanism for theinitiation of CIN and provides a clue to the nature of dominantmutations responsible for aneuploidy in human cancer.

The rapid emergence of mitotic defects and aneuploid cell populationstriggered by the fusion protein in normal human astrocytes, combinedwith the correction of aneuploidy after short inhibition of FGFR-TKactivity indicate that aneuploidy is a key event in tumor induction bythe FGFR-TACC gene fusions. Induction of aneuploidy per se isdetrimental to cellular fitness (Sheltzer and Amon, 2011). Full-blowntumorigenesis requires cooperation between aneuploidy and geneticlesions that confer growth advantage and protect cells against thedetrimental effects of aneuploidy (Coschi and Dick, 2012; Holland andCleveland, 2009; Weaver and Cleveland, 2009). Therefore, the potenttumor-initiating activity of FGFR-TACC fusions shows that the noveloncoproteins have growth-promoting signaling functions that complementthe loss of mitotic fidelity with ensuing karyotypic alterations(Sheltzer and Amon, 2011).

Targeted therapies against common genetic alterations in GBM have notchanged the dismal clinical outcome of the disease, most likely becausethey have systematically failed to eradicate the truly addictingoncoprotein activities of GBM. The highly specific anti-tumor effectsand the correction of aneuploidy precipitated by FGFR-TK inhibition ofFGFR-TACC-driven GBM provide a strong rationale for clinical trialsbased on FGFR inhibitors in patients harboring FGFR-TACC rearrangements.The computational gene fusion discovery pipeline reported here detectedother GBM cases in which FGFR family genes are implicated in additionalgene fusions beyond the FGFR-TACC rearrangements. Therefore, thefrequency of 3.1% is likely to be an underestimate of the target GBMpatient population that may benefit from FGFR-TK inhibition.

Experimental Procedures

Cell Culture and Isolation and Maintenance of GSCs.

Rat1A, mouse astrocytes Ink4A;Arf−/−, and human astrocytes were culturedin DMEM supplemented with 10% FBS. Isolation and culture of GSCs wasperformed as described (Carro et al., 2010). For treatment in vitro withPD173074, AZD4547 or BJG398, cells infected with vector control, FGFR3,TACC3, FGFR-TACC fusions or FGFR3-TACC3-K508M were seeded in 96-wellplates and treated with increasing concentrations of FGFR inhibitors.After 72-120 h, growth rate was measured using the3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)assay. Data were expressed as mean±SD. Proliferation rate in GSC-1123infected with FGFR3 shRNA lentivirus was determined by platingdissociated gliomaspheres at 2×10⁴ cells/well in twelve-well plates 5days after infection. The number of viable cells was determined bytrypan blue exclusion in triplicate cultures obtained from triplicateindependent infections. Cell number was scored every other day.

DNA, RNA Preparation, Genomic and Real-Time Quantitative PCR (qRT-PCR).

The validation of fusion transcripts was performed using both genomicand RT-PCR with forward and reverse primer combinations designed withinthe margins of the paired-end read sequences detected by RNA-seq. DNA,RNA preparation and qRT-PCR were performed as described (Carro et al.,2010; Zhao et al., 2008). To identify novel fusion transcripts withinthe GBM cohort, PCR primers pairs were designed to bind upstream to theTK domain of the FGFR genes and inside or downstream the Coiled Coildomain of the TACC genes. Expressed fusion transcript variants weresubjected to direct sequencing to confirm sequence and frame. Primersequences are included below.

Subcutaneous Xenografts and Drug Treatment.

Rat1A or Ink4A;Arf−/− astrocytes (5×10⁵) transduced with differentlentiviral constructs were suspended in 150 μl of PBS, together with 30μl of Matrigel (BD Biosciences), and injected subcutaneously in theflank of athymic nude (Nu/Nu) mice (Charles River Laboratories,Wilmington, Mass.). For experiments with FGFR inhibitors, mice carrying˜200-300 mm³ subcutaneous tumors derived from Ink4A;Arf−/− astrocyteswere randomized to receive 50 mg/kg PD173074 in 0.05 M lactate buffer(pH 5) or an equal volume of lactate buffer by oral gavage. Treatmentwas administered for three cycles consisting of four consecutive daysfollowed by two days of rest. Tumor diameters were measured withcaliper, and tumor volumes estimated using the formula:0.5×length×width. Data are expressed as mean±SE. Mice were sacrificedwhen tumors in the control group reached the maximal size allowed.

Orthotopic Transplantation and Drug Treatment.

Ink4A;Arf−/− astrocytes carrying a luciferase expressing vector weretransduced with FGFR3-TACC3 lentivitus. 1×10³ cells in 2 μl of salinewere injected in the caudate-putamen of 4-6 week old male athymic nude(Nu/Nu) mice using a stereotaxic frame (coordinates relative to bregma:0.5 mm anterior; 1.1 mm lateral; 3.0 mm ventral) and a 26 gauge Hamiltonsyringe. Six days after injection, mice underwent bioluminescenceimaging using a Xenogen CCD apparatus and were randomized to receive 50mg/kg AZD4547 in 1% Tween 80 (treatment group) or DMSO in an equalvolume of vehicle by oral gavage (control group). AZD4547 wasadministered daily for two cycles of 10 days with a two day interval.Mice were monitored daily and sacrificed when neurological symptomsappeared. Kaplan-Meier survival curve was generated using the DNAStatview software package (AbacusConcepts, Berkeley, Calif.). Log-rankanalysis was performed on the Kaplan-Meier survival curve to determinestatistical significance.

Intracranial Injections of Lentiviruses.

Intracranial injection of FGFR3-TACC3-shp53, EGFRvIII-shp53 or shp53pTomo lentiviruses was performed in 4 week-old C57/BL/6J mice inaccordance with guidelines of IACUC Committee. Briefly, 1.8 μl ofpurified lentiviral particles in PBS (1×10⁹/ml) were injected into thedentate gyms using a stereotaxic frame (coordinates relative to bregma:1.45 mm posterior; 1.65 mm lateral; 2.4 mm ventral) and a 26 gaugeHamilton syringe. Mice were monitored daily and sacrificed whenneurological symptoms appeared. Mouse brain was analyzedhistopathologically and by immunofluorescence staining.

Histology and Immunostaining.

Tissue preparation and immunohistochemistry on brain tumors andimmunofluorescence staining were performed as previously described(Carro et al., 2010; Zhao et al., 2009; Zhao et al., 2008). Antibodiesused in immunostaining and immunoblotting are listed below.

Cloning and Lentiviral Production.

Lentivirus preparation and infections were performed as described (Carroet al., 2010) and are detailed in Extended Experimental Procedures.

Karyotype Analysis.

Cultured cells were colcemid (20 ng/ml) treated for 90 minutes beforeharvesting for karyotopic analysis as detailed in Extended Experimentalprocedures. At least one hundred cells in metaphase were examined forchromosome count. PMSCS was scored in cells where a majority of thesister chromosomes were no longer associated. Two-tailed unpairedt-tests with Welch's correction were performed for comparison of meansanalysis.

Immunofluorescence and Live-Cell Microscopy.

Immunofluorescence microscopy was performed on cells fixed with 4% PFAin PHEM (60 mM Pipes, 27 mM Hepes, 10 mM EGTA, 4 mM MgSO₄, pH 7.0).Cells were permeabilized using 1% Triton X-100. Mitotic spindles werevisualized by anti-α-tubulin antibody (Sigma). Secondary antibodiesconjugated to Alexa Fluor-488/-594 (Molecular Probes) were used. Allstaining with multiple antibodies were performed in a sequential manner.DNA was stained by DAPI (Sigma). Fluorescence microscopy was performedon a Nikon A1R MP microscope.

Identification of Gene Fusions from Whole Transcriptome (RNA-Seq) andExome Sequencing.

RNA-Sequencing was performed from total RNA extracted from GSC culturesisolated from nine GBM patients using Illumina HiSeq 2000, producingroughly 60.3 million paired reads per sample. Using the global alignmentsoftware Burrows-Wheeler Aligner (BWA) (Li and Durbin, 2009) withmodified Mott's trimming, an initial seed length of 32, maximum editdistance of 2 and a maximum gap number of 1, on average 43.1 millionreads were mapped properly to the RefSeq transcriptome and, of theremaining, 8.6 million were mapped to the hg19 genome per sample. Theremaining 14.3% of paired reads—including those that failed to map toeither transcriptome or genome with proper forward-reverse (F-R)orientation, within expected insert size, and with minimal soft clipping(unmapped portions at the ends of a read)—were considered to beappropriate for gene fusion analysis.

A novel computational pipeline was constructed called TX-Fuse thatidentifies two sources of evidence for the presence of a gene fusion: 1.Split inserts, in which each read of a mate pair maps entirely to oneside of a breakpoint, and 2. Individual split reads that span abreakpoint. Split inserts are readily detected from BWA mapping. On theother hand, split reads demand precision alignment of smaller nucleotidestretches. To that end, the pipeline employs the local alignment packageBLAST with word size of 20, identity cutoff of 95%, expectation cutoffof 10⁻⁴, and soft filtering to map raw paired reads against the RefSeqtranscriptome. From this procedure, a list of potential split reads wereobtained that were filtered to ensure maintenance of coding frame in thepredicted fusion transcript given the proper F-R orientation in the readpair. False positive candidates produced from paralogous gene pairs werealso screened out using the Duplicated Genes Database and theEnsemblCompara GeneTrees (Vilella et al., 2009). Pseudogenes in thecandidate list were annotated using the list from HUGO Gene NomenclatureCommittee (HGNC) database (Seal et al., 2011) and given lower priority.For each remaining gene fusion candidate, a virtual reference wascreated based on the predicted fusion transcript and re-mapped allunmapped reads using BLAST with word size of 16, identity cutoff of 85%,query coverage greater than 85%, and expectation cutoff of 10⁻⁴ toobtain a final count of split reads and inserts. Moreover, sequencingdepth per base of the virtual reference was calculated to corroboratethat components of each gene participating in the gene fusion werehighly expressed.

To establish the recurrence of the initial panel of gene fusioncandidates, the gene fusion discovery pipeline was modified to produceEXome-Fuse, which probes for fusions within the available dataset ofpaired-read exome DNA sequencing of 84 matched GBM samples from TCGA. Toincrease sensitivity for gene fusion identification, reads unmapped byBWA were aligned to the gene pair participating in each fusion candidateusing a BLAST word size of 24 for split inserts and 16 for split readand split insert discovery. Given that the breakpoint detected in DNAcannot directly indicate the resulting breakpoint in the transcribedRNA, no restriction was made on split insert orientation. For splitreads, it was only required that the component of the split read mappedto the same gene as its mate maintained F-R directionality.

Co-Outlier Expression and CNV Analysis from TCGA GBM Samples.

Tomlins et al. (Tomlins et al., 2005) reported that outlier geneexpression from microarray datasets identifies candidate oncogenic genefusions. Wang et al. (Wang et al., 2009) suggested a “breakpointprinciple” for intragenic copy number aberrations in fusion partners.The two principles (outlier expression and intragenic CNV) were combinedto identify candidate gene fusions in GBM samples from Atlas-TCGA.Genomic and expression data sets were downloaded from TCGA public dataportal as available on Dec. 1, 2011, where a description of TCGA datatypes, platforms, and analyses is also available (2008). Specific datasources were (according to Data Levels and Data Types) as follows:Expression data, “Level 2” normalized signals per probe set (AffymetrixHT_HG-U133A) of 84 samples; Copy number data, “Level 1” raw signals perprobe (Affymetrix Genome-Wide Human SNP Array 6.0) of the 4 FGFR3-TACC3gene fusion positive samples (tumor and matched normal control).

The gene expression analysis was performed first using R³. The medianabsolute deviation (MAD) was calculated and then a gene was labeled asan outlier according to the following formula:Z_(i,j)=0.6745(x_(i,j)−mean(x_(i)))/MAD_(i)>3.5 (Iglewicz and Hoaglin,1993). Samples were identified as ECFS (expression candidate fusionsample) if both genes of interest (e. g. FGFR3 and TACC3) displayedoutlier behavior (co-outliers). Next, ECFS were analyzed for CNV usingpennCNV (Wang et al., 2007). Tumors samples were paired to their normalcontrols to obtain the log ratio values and the VEGA algorithm was usedto obtain a more accurate segmentation (Morganella et al., 2010).

Karyotypic Analysis.

The colcemid treated cells were trypsinized, centrifuged for 7 minutesat 200×g, and the cell pellet re-suspended in warmed hypotonic solutionand incubated at 37° C. for 13 minutes. The swollen cells were thencentrifuged and the pellet re-suspended in 8 ml of Carnoy's fixative(3:1 methanol:glacial acetic acid). The cell suspension was centrifugedand washed twice in Carnoy's fixative. After the last centrifugation,the cells were resuspended in 0.5 to 1 ml of freshly prepared fixativeto produce an opalescent cell suspension. Drops of the final cellsuspension were placed on clean slides and air-dried. Slides werestained with DAPI and metaphases were analyzed under a fluorescentmicroscope.

Cloning and Lentiviral Production.

Lentiviral expression vectors, pLOC-GFP (Open Biosystems) andpTomo-shp53, were used to clone FGFR3, TACC3, FGFR3-TACC3,FGFR3-TACC3-K508M, and FGFR1-TACC1. pTomo-shp53 was a gift of InderVerma and Dinorah Friedman-Morvinski (Salk Institute, San Diego). TheFGFR3-TACC3-K508M mutant was generated using the Phusion Site DirectMutagenesis kit (NEB, USA). MISSION shRNAs clones (pLKO.1 lentiviralexpression vectors) against FGFR3 were purchased from Sigma. The hairpinsequences targeting the FGFR3 gene are—

(#TRCN0000000372; Sh#2) (SEQ ID NO: 182) 5′-TGCGTCGTGGAGAACAAGTTT-3′;(#TRCN0000430673; Sh#3) (SEQ ID NO: 183) 5′-GTTCCACTGCAAGGTGTACAG-3′;(#TRCN0000000374; Sh#4) (SEQ ID NO: 184) 5′-GCACAACCTCGACTACTACAA-3′.

Genomic and mRNA RT-PCR.

Total RNA was extracted from cells by using RNeasy Mini Kit (QIAGEN),following the manufacturer instructions. 500 ng of total RNA wasretro-transcribed by using the Superscript III kit (Invitrogen),following the manufacturer instructions. The cDNAs obtained after theretro-transcription was used as templates for qPCR. The reaction wasperformed with a Roche480 thermal cycler, by using the Absolute BlueQPCR SYBR Green Mix from Thermo Scientific. The relative amount ofspecific mRNA was normalized to 18S. Results are presented as themean±SD of triplicate amplifications.

Primers used are:

hFGFR3-RT-FW1: (SEQ ID NO: 162) 5′-GTAACCTGCGGGAGTTTCTG-3′;hFGFR3-RT-REV1: (SEQ ID NO: 163) 5′-ACACCAGGTCCTTGAAGGTG-3′;hTACC3-RT-FW2:  (SEQ ID NO: 164) 5′-CCTGAGGGACAGTCCTGGTA-3′;hTACC3-RT-REV2:  (SEQ ID NO: 165) 5′-AGTGCTCCCAAGAAATCGAA-3′;hWRAP53-RT-FW1:  (SEQ ID NO: 180) 5′-AGAGGTGACCACCAATCAGC-3′;hWRAP53-RT-REV1:  (SEQ ID NO: 181) 5′-CGTGTCCCACACAGAGACAG-3′.

Primers used for the screening of FGFR-TACC fusions are:

FGFR3-FW1: (SEQ ID NO: 166) 5′-CGTGAAGATGCTGAAAGACGATG-3′; TACC3-REV1:(SEQ ID NO: 167) 5′- AAACGCTTGAAGAGGTCGGAG-3′; FGFR1-FW1:(SEQ ID NO: 168) 5′-ATGCTAGCAGGGGTCTCTGA-3′; TACC1-REV1:(SEQ ID NO: 169) 5′-CCCTTCCAGAACACCTTTCA-3′.

Primers used for genomic detection of FGFR3-TACC3 fusion in GBM-1123 andGSC-1123 are:

Genomic FGFR3-FW1: (SEQ ID NO: 170) 5′-ATGATCATGCGGGAGTGC-3′;genomic TACC3-REV1: (SEQ ID NO: 171) 5′-GGGGGTCGAACTTGAGGTAT-3′.

Primers used to validate fusions detected by RNA-seq are:

POLR2A-FW1:  (SEQ ID NO: 172) 5′-CGCAGGCTTTTTGTAGTGAG-3′; WRAP53-REV1: (SEQ ID NO: 173) 5′-TGTAGGCGCGAAAGGAAG-3′; PIGU-FW1:  (SEQ ID NO: 174)5′-GAACTCATCCGGACCCCTAT-3′; NCOA6-REV1:  (SEQ ID NO: 175)5′-GCTTTCCCCATTGCACTTTA-3′; ST8SIA4-FW1:  (SEQ ID NO: 176)5′-GAGGAGAGAAGCACGTGGAG-3′; PAM-REV1:  (SEQ ID NO: 177)5′-GGCAGACGTGTGAGGTGTAA-3′; CAPZB-FW:  (SEQ ID NO: 178)5′-GTGATCAGCAGCTGGACTGT-3′; UBR4-REV1:  (SEQ ID NO: 179)5′-GAGCCTGGGCATGGATCT-3′.

Confocal Microscopy Imaging.

For immunofluorescence of fixed cells, images were recorded with aZ-optical spacing of 0.25 μm using a Nikon AIR MP and a 60X1.3 oilobjective and analyzed using ImageJ software (National Institute ofHealth). For live-cell analyses, Rat1A cells infected with pLNCX-H2Bretrovirus and transduced with lentiviral vector or FGFR3-TACC3 fusionwere seeded in glass bottom dishes in phenol red free DMEM and followedby time-lapse microscopy using the Nikon AIR MP biostation at 37° C. and5% CO₂/95% air. Images with a Z-optical spacing of 1 μm were recordedevery 4 min for 8 h. Images of unchallenged mitosis from early prophaseuntil cytokinesis were processed using ImageJ software (NationalInstitute of Health). The time-point of nuclear envelope breakdown (NEB)was defined as the first frame showing loss of smooth appearance ofchromatin and anaphase was the first frame when chromosome movementtowards the poles became apparent. Nuclear envelope reconstitution (NER)was defined as the first frame showing nuclei decondensation.

Box and whisker plots were calculated from image sequences from at least50 recorded cells. Two-tailed unpaired t-tests with Welch's correctionwere performed for comparison of means analysis using StatView software(AbacusConcepts, Berkeley, Calif.).

Immunofluorescence.

Antibodies and concentrations used in immunofluorescence staining are:

Anti-Ki67 Rabbit 1:1000 Vector Labs Anti-pHH3 Rabbit 1:500  MilliporeAnti-FGFR3 Mouse 1:1000 Santa Cruz Anti-Tacc3 Goat 1:1000 USBiologicalAnti-a-tubulin Mouse 1:1000 Sigma Anti-Nestin Mouse 1:1000 BD PharmingenAnti-Olig2 Rabbit 1:200  IBL Anti-GFAP Rabbit 1:200  Dako Anti-ERKRabbit 1:1000 Cell Signaling Anti-pERK Rabbit 1:1000 Cell SignalingAntiFRS Rabbit 1:250  Santa Cruz Anti-pFRS Rabbit 1:1000 Cell SignalingAnti-AKT Rabbit 1:1000 Cell Signaling Anti-pAKT473 Rabbit 1:1000 CellSignaling

REFERENCES

-   Ablain, J., Nasr, R., Bazarbachi, A., and de The, H. (2011). The    Drug-Induced Degradation of Oncoproteins: An Unexpected Achilles'    Heel of Cancer Cells? Cancer Discov. 1, 117-127.-   Bass, A. J., Lawrence, M. S., Brace, L. E., Ramos, A. H., Drier, Y.,    Cibulskis, K., Sougnez, C., Voet, D., Saksena, G., Sivachenko, A.,    et al. (2011). Genomic sequencing of colorectal adenocarcinomas    identifies a recurrent VTI1A-TCF7L2 fusion. Nat. Genet. 43, 964-968.-   Cahill, D. P., Kinzler, K. W., Vogelstein, B., and Lengauer, C.    (1999). Genetic instability and darwinian selection in tumours.    Trends Cell. Biol. 9, M57-60.-   Carro, M. S., Lim, W. K., Alvarez, M. J., Bollo, R. J., Zhao, X.,    Snyder, E. Y., Sulman, E. P., Anne, S. L., Doetsch, F., Colman, H.,    et al. (2010). The transcriptional network for mesenchymal    transformation of brain tumours. Nature 463, 318-325.-   Coschi, C. H., and Dick, F. A. (2012). Chromosome instability and    deregulated proliferation: an unavoidable duo. Cell. Mol. Life Sci.    69, 2009-2024-   Druker, B. J. (2009). Perspectives on the development of imatinib    and the future of cancer research. Nat. Med. 15, 1149-1152.-   Furnari, F. B., Fenton, T., Bachoo, R. M., Mukasa, A., Stommel, J.    M., Stegh, A., Hahn, W. C., Ligon, K. L., Louis, D. N., Brennan, C.,    et al. (2007). Malignant astrocytic glioma: genetics, biology, and    paths to treatment. Genes Dev. 21, 2683-2710.-   Gavine, P. R., Mooney, L., Kilgour, E., Thomas, A. P., Al-Kadhimi,    K., Beck, S., Rooney, C., Coleman, T., Baker, D., Mellor, M. J., et    al. (2012). AZD4547: An Orally Bioavailable, Potent, and Selective    Inhibitor of the Fibroblast Growth Factor Receptor Tyrosine Kinase    Family. Cancer Res. 72, 2045-2056.-   Gerber, D. E., and Minna, J. D. (2010). ALK inhibition for non-small    cell lung cancer: from discovery to therapy in record time. Cancer    Cell 18, 548-551.-   Gordon, D. J., Resio, B., and Pellman, D. (2012). Causes and    consequences of aneuploidy in cancer. Nature reviews Genet. 13,    189-203.-   Guagnano, V., Furet, P., Spanka, C., Bordas, V., Le Douget, M.,    Stamm, C., Brueggen, J., Jensen, M. R., Schnell, C., Schmid, H., et    al. (2011). Discovery of    3-(2,6-dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-methyl-urea    (NVP-BGJ398), a potent and selective inhibitor of the fibroblast    growth factor receptor family of receptor tyrosine kinase. J. Med.    Chem. 54, 7066-7083.-   Holland, A. J., and Cleveland, D. W. (2009). Boveri revisited:    chromosomal instability, aneuploidy and tumorigenesis. Nat. Rev.    Mol. Cell. Biol. 10, 478-487.-   Hood, F. E., and Royle, S. J. (2011). Pulling it together: The    mitotic function of TACC3. Bioarchitecture 1, 105-109.-   Lee, J., Kotliarova, S., Kotliarov, Y., Li, A., Su, Q., Donin, N.    M., Pastorino, S., Purow, B. W., Christopher, N., Zhang, W., et al.    (2006). Tumor stem cells derived from glioblastomas cultured in bFGF    and EGF more closely mirror the phenotype and genotype of primary    tumors than do serum-cultured cell lines. Cancer Cell 9, 391-403.-   Lengauer, C., Kinzler, K. W., and Vogelstein, B. (1997). Genetic    instability in colorectal cancers. Nature 386, 623-627.-   Lengauer, C., Kinzler, K. W., and Vogelstein, B. (1998). Genetic    instabilities in human cancers. Nature 396, 643-649.-   Lo, H. W. (2010). EGFR-targeted therapy in malignant glioma: novel    aspects and mechanisms of drug resistance. Curr. Mol. Pharmacol. 3,    37-52.-   Marumoto, T., Tashiro, A., Friedmann-Morvinski, D., Scadeng, M.,    Soda, Y., Gage, F. H., and Verma, I. M. (2009). Development of a    novel mouse glioma model using lentiviral vectors. Nat. Med. 15,    110-116.-   Mitelman, F., Johansson, B., and Mertens, F. (2007). The impact of    translocations and gene fusions on cancer causation. Nat. Rev.    Cancer 7, 233-245.-   Mohammadi, M., Froum, S., Hamby, J. M., Schroeder, M. C., Panek, R.    L., Lu, G. H., Eliseenkova, A. V., Green, D., Schlessinger, J., and    Hubbard, S. R. (1998). Crystal structure of an angiogenesis    inhibitor bound to the FGF receptor tyrosine kinase domain. EMBO J.    17, 5896-5904.-   Ohgaki, H., and Kleihues, P. (2005). Population-based studies on    incidence, survival rates, and genetic alterations in astrocytic and    oligodendroglial gliomas. J. Neuropathol. Exp. Neurol. 64, 479-489.-   Peset, I., and Vernos, I. (2008). The TACC proteins: TACC-ling    microtubule dynamics and centrosome function. Trends Cell. Biol. 18,    379-388.-   Prensner, J. R., and Chinnaiyan, A. M. (2009). Oncogenic gene    fusions in epithelial carcinomas. Curr Opin Genet. Dev. 19, 82-91.-   Reardon, D. A., Desjardins, A., Vredenburgh, J. J., Gururangan, S.,    Friedman, A. H., Herndon, J. E., 2nd, Marcello, J., Norfleet, J. A.,    McLendon, R. E., Sampson, J. H., et al. (2010). Phase 2 trial of    erlotinib plus sirolimus in adults with recurrent glioblastoma. J.    Neurooncol. 96, 219-230.-   Sheltzer, J. M., and Amon, A. (2011). The aneuploidy paradox: costs    and benefits of an incorrect karyotype. Trends Genet. 27, 446-453.-   Solomon, D. A., Kim, T., Diaz-Martinez, L. A., Fair, J.,    Elkahloun, A. G., Harris, B. T., Toretsky, J. A., Rosenberg, S. A.,    Shukla, N., Ladanyi, M., et al. (2011). Mutational inactivation of    STAG2 causes aneuploidy in human cancer. Science 333, 1039-1043.-   Stephens, P. J., McBride, D. J., Lin, M. L., Varela, I.,    Pleasance, E. D., Simpson, J. T., Stebbings, L. A., Leroy, C.,    Edkins, S., Mudie, L. J., et al. (2009). Complex landscapes of    somatic rearrangement in human breast cancer genomes. Nature 462,    1005-1010.-   Still, I. H., Vince, P., and Cowell, J. K. (1999). The third member    of the transforming acidic coiled coil-containing gene family,    TACC3, maps in 4p16, close to translocation breakpoints in multiple    myeloma, and is upregulated in various cancer cell lines. Genomics    58, 165-170.-   Thompson, S. L., Bakhoum, S. F., and Compton, D. A. (2010).    Mechanisms of chromosomal instability. Curr. Biol. 20, R285-295.-   Tomlins, S. A., Laxman, B., Dhanasekaran, S. M., Helgeson, B. E.,    Cao, X., Morris, D. S., Menon, A., Jing, X., Cao, Q., Han, B., et    al. (2007). Distinct classes of chromosomal rearrangements create    oncogenic ETS gene fusions in prostate cancer. Nature 448, 595-599.-   Tomlins, S. A., Rhodes, D. R., Perner, S., Dhanasekaran, S. M.,    Mehra, R., Sun, X. W., Varambally, S., Cao, X., Tchinda, J., Kuefer,    R., et al. (2005). Recurrent fusion of TMPRSS2 and ETS transcription    factor genes in prostate cancer. Science 310, 644-648.-   Turner, N., and Grose, R. (2010). Fibroblast growth factor    signalling: from development to cancer. Nat. Rev. Cancer 10,    116-129.-   van den Bent, M. J., Brandes, A. A., Rampling, R., Kouwenhoven, M.    C., Kros, J. M., Carpentier, A. F., Clement, P. M., Frenay, M.,    Campone, M., Baurain, J. F., et al. (2009). Randomized phase II    trial of erlotinib versus temozolomide or carmustine in recurrent    glioblastoma: EORTC brain tumor group study 26034. J. Clin. Oncol.    27, 1268-1274.-   Wang, X. S., Prensner, J. R., Chen, G., Cao, Q., Han, B.,    Dhanasekaran, S. M., Ponnala, R., Cao, X., Varambally, S.,    Thomas, D. G., et al. (2009). An integrative approach to reveal    driver gene fusions from paired-end sequencing data in cancer. Nat.    Biotechnol. 27, 1005-1011.-   Weaver, B. A., and Cleveland, D. W. (2009). The role of aneuploidy    in promoting and suppressing tumors. J. Cell. Biol. 185, 935-937.-   Wesche, J., Haglund, K., and Haugsten, E. M. (2011). Fibroblast    growth factors and their receptors in cancer. Biochem. J. 437,    199-213.-   Yan, H., Parsons, D. W., Jin, G., McLendon, R., Rasheed, B. A.,    Yuan, W., Kos, I., Batinic-Haberle, I., Jones, S., Riggins, G. J.,    et al. (2009). IDH1 and IDH2 mutations in gliomas. New Engl. J. Med.    360, 765-773.-   Zhao, X., D, D. A., Lim, W. K., Brahmachary, M., Carro, M. S.,    Ludwig, T., Cardo, C. C., Guillemot, F., Aldape, K., Califano, A.,    et al. (2009). The N-Myc-DLL3 cascade is suppressed by the ubiquitin    ligase Huwel to inhibit proliferation and promote neurogenesis in    the developing brain. Dev. Cell 17, 210-221.-   Zhao, X., Heng, J. I., Guardavaccaro, D., Jiang, R., Pagano, M.,    Guillemot, F., Iavarone, A., and Lasorella, A. (2008). The    HECT-domain ubiquitin ligase Huwel controls neural differentiation    and proliferation by destabilizing the N-Myc oncoprotein. Nat Cell    Biol 10, 643-653.-   (2008). Comprehensive genomic characterization defines human    glioblastoma genes and core pathways. Nature 455, 1061-1068.-   Iglewicz, B., and Hoaglin, D. C. (1993). How to detect and handle    outliers (Milwaukee, Wis.: ASQC).-   Li, H., and Durbin, R. (2009). Fast and accurate short read    alignment with Burrows-Wheeler transform. Bioinformatics 25,    1754-1760.-   Morganella, S., Cerulo, L., Viglietto, G., and Ceccarelli, M.    (2010). VEGA: variational segmentation for copy number detection.    Bioinformatics 26, 3020-3027.-   Seal, R. L., Gordon, S. M., Lush, M. J., Wright, M. W., and    Bruford, E. A. (2011). genenames.org: the HGNC resources in 2011.    Nucleic Acids Res 39, D514-519.-   Tomlins, S. A., Rhodes, D. R., Perner, S., Dhanasekaran, S. M.,    Mehra, R., Sun, X. W., Varambally, S., Cao, X., Tchinda, J., Kuefer,    R., et al. (2005). Recurrent fusion of TMPRSS2 and ETS transcription    factor genes in prostate cancer. Science 310, 644-648.-   Vilella, A. J., Severin, J., Ureta-Vidal, A., Heng, L., Durbin, R.,    and Birney, E. (2009). EnsemblCompara GeneTrees: Complete,    duplication-aware phylogenetic trees in vertebrates. Genome Res. 19,    327-335.-   Wang, K., Li, M., Hadley, D., Liu, R., Glessner, J., Grant, S. F.,    Hakonarson, H., and Bucan, M. (2007). PennCNV: an integrated hidden    Markov model designed for high-resolution copy number variation    detection in whole-genome SNP genotyping data. Genome Res. 17,    1665-1674.-   Wang, X. S., Prensner, J. R., Chen, G., Cao, Q., Han, B.,    Dhanasekaran, S. M., Ponnala, R., Cao, X., Varambally, S.,    Thomas, D. G., et al. (2009). An integrative approach to reveal    driver gene fusions from paired-end sequencing data in cancer. Nat.    Biotechnol. 27, 1005-1011.

Example 2 Fusions in GBM

TABLE 8 Soft agar colony assay Cell line Vector FGFR3 TACC3 F1-T1 FusionF3-T3 Fusion F3-T3-K508M Fusion Rat1 0 0 0 225.3 ± 10.0 198.7 ± 8.0 0Balb 3T3 0 0 0 n.d.  45.5 ± 8.9 n.d. n.d.: not done

TABLE 9 Subcutaneous tumor xenografts Cell line Vector FGFR3 TACC3 F1-T1Fusion F3-T3 Fusion F3-T3-K508M Fusion Rat1 0/5 0/5 0/5 n.d. 5/5 n.d.Ink4A/Arf−/− 0/9 0/5 0/5 8/8 12/12 0/8 Astrocytes n.d.: not done

TABLE 10 Analysis of chromosomal number in Rat1 cells Number of cellsPercent Mean Average variation Cell line counted aneuploidy Range numberfrom mean number p-value Rat1A Vector 100 27 35-43 41.2 1.2 Rat1A FGFR3100 33 35-44 42.1 1.3 n.s. Rat1A TACC3 100 41 34-46 40.7 1.1 n.s. Rat1AFGFR3-TACC3 100 69 35-73 43.6 3.1 <0.0001

TABLE 11 Analysis of chromosomal number in human astrocytes Number ofPercent Mean Average variation Cell line cells counted aneuploidy Rangenumber from mean number p-value Human Astrocytes Vector 100 8 42-4645.85 0.28 p = <0.001 Human Astrocytes FGFR3-TACC3 100 42 28-48 42.243.33

Example 3 Fusions in Other Cancers

The inventors previously reported in Example 1 that 3.1% of humanglioblastoma harbor FGFR3-TACC3 and FGFR1-TACC1 gene fusions. Tumorsharboring FGFR3-TACC3 gene fusions are identified by the presence ofhighly specific focal micro-amplification events of the rearrangedportions of the FGFR3 and TACC3 genes (See FIG. 2E). Therefore, thesemicro-amplification events can be used as distinctive marks for thepresence of FGFR3-TACC3 gene fusions. It was asked whether other typesof human tumors also harbor FGFR3-TACC3 gene fusions from the analysisof Copy Number Variations (CNVs) of SNP arrays generated from theAtlas-TCGA project. This analysis was performed using segmented CNVsdata visualized using the Integrated Genomic Viewers software. Theanalysis revealed that the following tumors, shown in the FIGS. 31-35,display focal micro-amplification events of FGFR3 and TACC3 thatindicate the presence of FGFR3-TACC3 gene fusions (in FIGS. 31-35, redindicates amplification (A), blue indicates deletion (D); FIG. 31:Bladder Urothelial Carcinoma; FIG. 32: Breast Carcinoma; FIG. 33:Colorectal Carcinoma; FIG. 34: Lung Squamous Cell Carcinoma; FIG. 35:Head and Neck Squamous Cell Carcinoma).

Taken together, these data indicate that the same FGFR3-TACC3 genefusions reported for the first time in Glioblastoma also occur inseveral other types of human tumors. Therefore, as for Glioblastoma andother epithelial cancers (such as the human tumors discussed herein),the identification of FGFR-TACC gene fusions also provides a newdiagnostic and therapeutic target for treatment with drugs that inhibitFGFR-TACC gene fusions.

Example 4 Detection, Characterization and Inhibition of FGFR-TACCFusions in IDH Wild Type Glioma

Translational Relevance

Described herein is an unbiased screening assay for FGFR-TACC fusions inglioma that overcomes the great variability of variants that aregenerated by FGFR-TACC chromosomal translocation in human cancer.FGFR-TACC fusions occur in grade II and III glioma harboring wildtypeIDH1 with frequency similar to glioblastoma (GBM), therefore providing aclue to the aggressive clinical behavior of this glioma subtype. Themolecular characterization of fusion-positive glioma revealed thatFGFR-TACC is mutually exclusive with EGFR amplification but co-occurswith CDK4 amplification. FGFR-TACC-positive glioma displays strikinglyuniform and strong expression of the fusion protein at the single celllevel. Preclinical experiments with FGFR3-TACC3-positive glioma cellstreated with the FGFR inhibitor JNJ-42756493 showed strong antitumoreffects and treatment of two patients with recurrent GBM harboringFGFR3-TACC3 resulted in clinical improvement and radiological tumorreduction. These findings validate the treatment with FGFR inhibitors ofglioma patients harboring FGFR-TACC chromosomal translocations.

Abstract

Purpose.

Oncogenic fusions consisting of FGFR and TACC are present in a subgroupof glioblastoma (GBM) and other human cancers and have been proposed asnew therapeutic targets. Frequency, molecular features of FGFR-TACCfusions, and the therapeutic efficacy of inhibiting FGFR kinase in GBMand grade-II-III glioma were analyzed.

Experimental Design.

Overall, 795 gliomas (584 GBM, 85 grade-II-III with wild-type and 126with IDH1/2 mutation) were screened for FGFR-TACC breakpoints andassociated molecular profile. Expression of the FGFR3 and TACC3components of the fusions were also analyzed. The effects of thespecific FGFR inhibitor JNJ-42756493 for FGFR3-TACC3-positive gliomawere determined in preclinical experiments. Two patients with advancedFGFR3-TACC3-positive GBM received JNJ-42756493 and were assessed fortherapeutic response.

Results.

Three of 85 IDH1/2 wild type (3.5%) but none of 126 IDH1/2 mutantgrade-II-III glioma harbored FGFR3-TACC3 fusions. FGFR-TACCrearrangements were present in 17 of 584 GBM (2.9%). FGFR3-TACC3 fusionswere associated with strong and homogeneous FGFR3 immunostaining. Theyare mutually exclusive with IDH1/2 mutations and EGFR amplificationwhereas co-occur with CDK4 amplification. JNJ-42756493 inhibited growthof glioma cells harboring FGFR3-TACC3 in vitro and in vivo. The twopatients with FGFR3-TACC3 rearrangements who received JNJ-42756493manifested clinical improvement with stable disease and minor response,respectively.

Conclusions.

RT-PCR-sequencing is a sensitive and specific method to identifyFGFR-TACC-positive patients. FGFR3-TACC3 fusions are associated withuniform intra-tumor expression of the fusion protein. The clinicalresponse observed in the FGFR3-TACC3-positive patients treated with aFGFR inhibitor supports clinical studies of FGFR inhibition inFGFR-TACC-positive patients.

Introduction

The history of successful targeted therapy of cancer largely coincideswith the inactivation of recurrent, oncogenic and addicting gene fusionsin hematological malignancies and recently in some types of epithelialcancer (1, 2). Glioblastoma multiforme (GBM) is among the most lethalforms of human cancer and targeted therapies against common geneticalterations in GBM have not changed the dismal outcome of the disease(3, 4). Underlying biological features including infiltrative growthbehavior, intratumoral heterogeneity, and adaptive resistance mechanismscoupled with the unique challenges of intracranial location presentsignificant problems in its effective management. Despite surgery andchemo-radiotherapy, most patients rapidly recur and no effectivetreatment options are available at that stage. Beside GBM, whichfeatures the highest grade of malignancy among glioma (grade IV), lowergrade glioma which include grade II and grade III are a heterogeneousgroup of tumors in which specific molecular features are associated withdivergent clinical outcome. The majority of grade II-III glioma (butonly a small subgroup of GBM) harbor mutations in IDH genes (IDH1 orIDH2), which confer a more favorable clinical outcome. Conversely, theabsence of IDH mutations is associated with the worst prognosis (5).

Described herein is the identification of FGFR-TACC gene fusions (mostlyFGFR3-TACC3, and rarely FGFR1-TACC1) as the first example of highlyoncogenic and recurrent gene fusions in GBM. The FGFR-TACC fusions thathave been identified so far include the Tyrosine Kinase (TK) domain ofFGFR and the coiled-coil domain of TACC proteins, both necessary for theoncogenic function of FGFR-TACC fusions. Tumor dependency on FGFR-TACCfusions was also tested in preclinical mouse models of FGFR-TACC gliomaand observed marked anti-tumor effects by FGFR inhibition (6).FGFR3-TACC3 fusions have been identified in pediatric and adult glioma,bladder carcinoma, squamous lung carcinoma and head and neck carcinoma,thus establishing FGFR-TACC fusions as one of the chromosomaltranslocation most frequently found across multiple types of humancancers (6-15).

From a mechanistic standpoint, the unexpected capacity of FGFR-TACCfusions to trigger aberrant chromosome segregation during mitosis, thusinitiating chromosome instability (CIN) and aneuploidy, two hallmarks ofcancer, is described herein. However, the full repertoire of thestructural variants of FGFR-TACC fusions occurring in GBM and lowergrade glioma is not completely understood. Furthermore, it remainsunknown whether FGFR-TACC fusions mark distinct grades of glioma and GBMsubtypes.

To date eight variants of the FGFR3-TACC3 fusion have been reported thatmostly differ for the breakpoint in the TACC3 gene (6-15). Because ofthe close proximity of FGFR3 and TACC3 (the two genes map at a distanceof 70 Kb on chromosome 4p16.3), detection of FGFR3-TACC3 rearrangementsby FISH is not a feasible option with the currently available methods.Here a screening method for FGFR-TACC fusions is reported that includesa RT-PCR assay designed to identify the known and novel FGFR3-TACC3fusion transcripts, followed by confirmation of the inframe breakpointby Sanger sequencing. Using this assay, a dataset of 584 GBM and 211grade II and grade III gliomas has been analyzed.

A crucial question with fundamental clinical relevance for any novelcandidate target mutation is the frequency of the alteration in thecancer cell population, thus discriminating between a clonal orsub-clonal origin of the mutation. In fact, GBM is characterized by aformidable degree of subclonal heterogeneity, whereby neighboring cellsdisplay amplification and expression of different Receptor TyrosineKinase (RTK)-coding genes (16-19). This notion poses major therapeuticchallenges for targeting any individual RTK will result, at best, in theeradication of a limited tumor sub-clone. Described herein, it wasdetermined that brain tumors harboring FGFR-TACC fusions manifest strongand homogeneous intra-tumor expression of the FGFR3 and TACC3 componentinvariably included in the fusion protein, when analyzed byimmunostaining. A significant clinical benefit following treatment witha specific inhibitor of FGFR-TK is reported in two GBM patients whoharbored FGFR3-TACC3 rearrangement.

Materials and Methods

Patients and Tissue Samples.

This example includes a cohort of 746 untreated patients with histologicdiagnosis of glioma from 5 institutions. Forty-nine recurrent gliomasfrom Pitié-Salpêtrière Hospital and one recurrent glioma from theUniversity of Calgary were also included. A summary of the patientcohort is provided in Table 12.

TABLE 12 Frequency of FGFR3-TACC3 Fusions in GBM and Grade II-IIIglioma. Distribution of the FGFR3-TACC3 fusions in GBM (upper panel) andlower grade glioma (lower panel) samples stratified according to theInstitution of origin. The table reports number of cases analyzed,number of tumors harboring FGFR3-TACC3 fusion transcripts, and resultsof FGFR3 immunostaining. Lower grade glioma samples are furtherclassified according to IDH status (IDH1 and IDH2). The respectivefrequency of FGFR3-TACC3 in GBM, Glioma grade II-III IDH wild type (wt),and IDH mutant (Mut) glioma is reported in parentheses. ImmunostainingNo of case No of detected FGFR3 positive/Sample Tumor sample source(GBM) fusions analyzed Pitié-Salpêtrière Hospital 380  9 9/9 BestaNeurological Institute 85 5 2/2 University of Calgary 60 + 1R^(§) 2 +1R^(§) 1/1 + 1/1R^(§) Montreal Neurological Institute 51 1 — Universityof British Columbia  8 0 — Total 584 (100%)^(£) 17 (2.9%) ImmunostainingNo of cases No of detected FGFR3 positive/Sample Tumor sample source IDHStatus (Grade II-III) fusions analyzed Pitié-Salpêtrière Hospital IDH wt85* (100%) 3 (3.5%) 3/3 IDH1/IDH2 Mut 126 (100%) 0 (0%)  0R^(§)Recurrent GBM. ^(£)Recurrent GBM from the University of CalgaryDataset is not included in the total count of GBM. *25 cases out of 85are unknown for IDH2 status.

Tumor specimens, blood samples and clinico-pathological information werecollected with informed consent and relevant ethical board approval inaccordance with the tenets of the Declaration of Helsinki. For thesamples from the Pitié-Salpêtrière Hospital, clinical data and follow-upare available in the neuro-oncology database (Onconeurotek, GHPitié-Salpêtrière, Paris).

Two recurrent GBM patients harboring FGFR3-TACC3 were enrolled in thedose escalation part of JNJ-42756493 trial at the Gustave RoussyInstitute.

Identification of Fusion Transcripts and Analysis of GenomicBreakpoints.

Total RNA was extracted from frozen tissues using Trizol (Invitrogen)according to manufacturer instructions. Two to three hundred nanogramsof total RNA were retro-transcribed with the Maxima First Strand cDNASynthesis Kit (Thermo Scientific) or SuperScript II (Invitrogen). RT-PCRwas performed using AccuPrime Taq DNA Polymerase (Invitrogen). Primerpairs used for the FGFR3-TACC3 fusions screening were: FGFR3ex12-FW:5′-CGTGAAGATGCTGAAAGACGATG-3 (SEQ ID NO: 495) and TACC3ex14-RV:5′-AAACGCTTGAAGAGGTCGGAG-3 (SEQ ID NO: 496); amplification conditionswere 94° C.-3 min, (94° C.-30 sec/61° C.-30 sec/68° C.-1 min 40 sec) for35 cycles, 68° C.-7 min. FGFR1-TACC1 fusions were amplified withFGFR1ex16-FW: 5′-TGCCTGTGGAGGAACTTTTCA-3′ (SEQ ID NO: 497) andTACC1ex13-RV: 5′-CCCAAACTCAGCAGCCTAAG-3′ (SEQ ID NO: 498) primers (94°C.-30 sec/60° C.-30 sec/68° C.-1 min 40 sec for 35 cycles). PCR productswere subjected to Sanger sequencing.

FGFR3-TACC3 genomic breakpoints were analyzed in 6 FGFR3-TACC3 positivesamples, 5 of which from the Pitié-Salpêtrière Hospital and 1 fromMontreal Neurological Institute. Three additional samples (MB-22, TCGA27-1835 and TCGA 06-6390) available from the previous study (6) werealso included in the analysis. Fifty nanograms of genomic DNA were usedin the PCR reaction, performed with Accuprime Taq Polymerase(Invitrogen) and PCR products were Sanger sequenced. Primers used ingenomic PCR were designed according to the breakpoint sequence in themRNA; the list of primers used are: FGFR3ex17-FW5′-TGGACCGTGTCCTTACCGT-3′ (SEQ ID NO: 499) (PCR Samples 3048, 4373,4867, 4451, MB-22, OPK-14, 06-6390, 27-1835 and Sequencing samples 3048,4373, 4867, 4451, MB-22, OPK14, 06-6390, 27-1835); FGFR3ex16-FW5′-GGTCCTTTGGGGTCCTGCT-3′ (SEQ ID NO: 500) (PCR and Sequencing Sample3808); TACC3ex6-RV 5′-CCTCTTTCAGCTCCAAGGCA-3′ (SEQ ID NO: 501) (PCR andSequencing Samples PCR 4451 and OPK-14); TACC3ex8-RV5′-TCTACCAGGACTGTCCCTCAG-3′ (SEQ ID NO: 502) (Sequencing Samples 3048and 4373); TACC3ex9-RV 5′-GGGAGTCTCATTTGCACCGT-3′ (SEQ ID NO: 503) (PCRSamples 3048, 4373, 4867 and Sequencing Sample 4867); TACC3ex10-RV5′-CTGCATCCAGGTCCTTCTGG-3′ (SEQ ID NO: 504) (PCR and Sequencing SamplesMB-22 and 06-6390); TACC3ex11-RV 5′-CCAGTTCCAGGTTCTTCCCG-3′ (SEQ ID NO:505) (Sequencing Samples 27-1837 and 3808); TACC3ex12-RV5′-CAACCTCTTCGAACCTGTCCA-3′ (SEQ ID NO: 506) (PCR and Sequencing Samples27-1837 and 3808). PCR conditions were 94° C.-30 sec/60° C.-30 sec/68°C.-2 min 30 sec for 40 cycles. For amplifications performed with theprimer TACC3ex9-RV, the program was 94° C.-30 sec/56° C.-30 sec/68° C.-2min 30 sec) for 40 cycles.

Quantitation of FGFR3 and TACC3 Transcripts in GBM.

The relative expression of FGFR3 and TACC3 regions included in orexcluded from the fusion transcript was assessed by qRT-PCR. Primerpairs with comparable efficiency of amplification were identified andefficiency was assessed using serial dilutions of cDNA (20) preparedfrom OAW28 ovarian carcinoma cells that contain wild type FGFR3 andTACC3 (21). Primers used are: N-terminal region of FGFR3, FGFR3-N:Forward 5′-AAGACGATGCCACTGACAAG-3′ (SEQ ID NO: 507), Reverse5′-CCCAGCAGGTTGATGATGTTTTTG-3′ (SEQ ID NO: 508); C-terminal region ofTACC3, TACC3-C: Forward 5′-TCCTTCTCCGACCTCTTCAAGC-3′ (SEQ ID NO: 509),Reverse 5′-TAATCCTCCACGCACTTCTTCAG-3′ (SEQ ID NO: 510). To amplifytranscripts in regions excluded from FGFR3-TACC3 fusion, primers weredesigned in the C-terminal region of FGFR3, FGFR3-C: Forward5′-TACCTGGACCTGTCGGCG-3′ (SEQ ID NO: 511), Reverse5′-TGGGCAAACACGGAGTCG-3′ (SEQ ID NO: 512) and N-terminal domain ofTACC3, TACC3-N: Forward 5′-CCACAGACGCACAGGATTCTAAGTC-3′ (SEQ ID NO:513), Reverse 5′-TGAGTTTTCCAGTCCAAGGGTG-3′ (SEQ ID NO: 514). Allreactions were performed in triplicate and the data are reported as FoldChange±Standard Deviation.

Immunofluorescence and Immunohistochemistry.

For immunofluorescence (IF) staining of FGFR3, 5 μm FFPE sectionssubjected to antigen retrieval with citrate buffer for 8 min. Primaryantibodies were: FGFR3-N (1:400, sc-13121, Santa Cruz Biotechnology),FGFR3-C (1:2000, sc-123, Santa Cruz Biotechnology), TACC3-N (1:600,ab134153, Abcam), and TACC3-C (1:300, NBP1-01032, Novus Biological).Secondary biotinylated antibodies were used at 1:50,000 followed bystreptavidin and TSA Cy3-conjugated. Nuclei were counterstained withDAPI. For immunohistochemical analysis (IHC) of FGFR3 expression,antigen retrieval was performed for 12 min and FGFR-3 antibody(sc-13121, Santa Cruz Biotechnology) was diluted 1:500. Biotinylatedanti-mouse antibody (1:30,000) and streptavidin were added beforeincubation with the chromogen. Nuclei were counterstaining withhematoxylin.

Molecular Characterization of Tumor Samples.

Mutational status of IDH1, IDH2, TERT promoter, as well as themethylation status of the MGMT promoter was analyzed in thePitié-Salpêtrière cohort. Expression of IDH1-R132H mutant was analyzedby IHC in 500 cases as previously described (22). IDH1 and IDH2 genemutations were identified by Sanger sequencing in 464 and 388 gliomas,respectively (5). IDH wild-type tumors are defined according to theabsence of IDH1-R132H immunopositivity and/or mutations in IDH1 and IDH2genes. TERT promoter status was determined by the same technique in 277samples (23). Hyper-methylation of the MGMT promoter was tested in 242samples by bisulfate pyro-sequencing (24). The presence of EGFRvIII wasevaluated by RT-PCR in 118 samples using EGFR-FW5′-CTTCGGGGAGCAGCGATGCGAC-3′ (SEQ ID NO: 515) and EGFR-RV5′CTGTCCATCCAGAGG AGGAGTA-3′ (SEQ ID NO: 516) primers (25).

Copy number variations analyses have been performed on 192 tissuesamples using CGH arrays using BAC arrays (N=187), Agilent 4x180K (N=2),Nimblegen 3x720K (N=2), Agilent 8x60K (N=1). Results were normalizedusing control DNA from matched blood samples as previously described(26). Additional analyses of 193 tumor specimens were performed by SNParray, using Illumina Omni (N=110), Illumina HumCore (N=32), Illumina370K (N=27), or Illumina 610K (N=24), as previously described (27).Array processing was outsourced to Integragen. Raw copy numbers wereestimated at each of the SNP and copy-number markers. Biodiscoveryproperty SNP-FASST2 algorithm was then used to segment copy number data.Segments were mapped to hg18 genome assembly (28). Copy numberalterations (CAN) magnitudes called log-R ratio (LRR) were classifiedusing simple thresholds: deletion (x≦−1), loss (−1<x≦−0.2), gain(0.2≦x<1) or amplification (x≧1) according to default Nexus 7.5software. For additional 56 gliomas, 10q loss was assessed on tumor andblood DNA by microsatellite analysis, while amplification of EGFR, MDM2and CDK4, and deletion of CDKN2A gene, were determined by qPCR, aspreviously reported (29, 30).

The molecular profiles obtained in Pitié-Salpêtrière dataset werecombined with those available in the TCGA dataportal. TCGA GBM segmentedcopy number variation profile was downloaded from The UCSC CancerGenomics Browser (31). Copy Number Variations (CNVs) were measuredexperimentally using the Affymetrix Genome-Wide Human SNP Array 6.0platform at the Broad TCGA genome characterization center (32). Raw copynumbers were estimated at each of the SNP and copy-number markers.Circular binary segmentation was then used to segment the copy numberdata (28). Segments are mapped to hg18 genome assembly at Broad.

For CNV analysis of the regions across FGFR3 and TACC3 genes, samplesfor which RNAseq and CNV data were available or samples for which onlyCNV data were available and RT-PCR-sequencing of FGFR3-TACC3 fusion hadbeen performed were considered. Overall, 158 GBM (all with a wild typeIDH1 gene) satisfied these criteria. Among them, 5 harbored anFGFR3-TACC3 fusion whereas 153 were FGFR-TACC-negative. The CNVmagnitudes, called log-R ratio (LRR), were classified using thefollowing thresholds: deletion (x<−1), loss (−1≦x≦−0.2), gain (0.2≦x≦1)or amplification (x>1), according to the Atlas-TCGA (32). The analysisof the genomic regions encompassing EGFR, MDM2, CDK4, CDKN2A, 7p, 10q,according to hg18 genome assembly, was performed to evaluate their CNV.EGFRvIII mutation status was inferred according to Brennan et al. (32).The frequencies of the aberrations of these genes in FGFR3-TACC3positive and negative samples were calculated and the obtained data werethen combined with the Pitié-Salpêtrière Hospital dataset.

Statistical Analysis.

Differences in the distribution on categorical variables were analyzedusing Fisher Exact test. The p-values were adjusted for multiple testingaccording to Benjamini and Hochberg false discovery rate (FDR). Aq-value≦0.05 (two-sided) was considered to be statistically significant.

Overall survival (OS) was defined as the time between the diagnosis anddeath or last follow-up. Patients who were still alive at the lastfollow-up were considered as censored events in the analysis.Progression-free survival (PFS) was defined as the time between thediagnosis and recurrence or last follow-up. Patients who wererecurrence-free at the last follow-up were considered as censored eventsin the analysis. Survival curves were calculated by the Kaplan-Meiermethod and differences between curves assessed using the Log-Rank test.A Log-Rank test p-value≦0.05 (two-sided) was considered to bestatistically significant.

Cell Culture and Cell Growth Assay.

GIC-1123 gliomaspheres were cultured in neurobasal medium (Invitrogen)supplemented with B27, N2 (Invitrogen), EGF and FGF2 (20 ng/ml,PeproTech). Mouse astrocytes Ink4A-Arf−/− were cultured in DMEMsupplemented with 10% Fetal Bovine Serum. Cells were seeded at 1,000cells/well in a 96-well plate and treated with JNJ-42756493. After 72hours cell viability was assessed using the3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)assay. Data are mean±SEM of six replicates. Experiments were performedthree times.

Subcutaneous Xenografts and Drug Treatment.

GIC-1123 cells (5×10⁵) were injected subcutaneously in the flank ofathymic nude (Nu/Nu) mice (Charles River Laboratories). Mice carrying˜200 mm³ subcutaneous tumors were randomized to receive 12 mg/kgJNJ-42756493 or DMSO in 1% Tween 80 by oral gavage. Tumor diameters weremeasured with caliper and tumor volumes estimated using the formula:0.5×length×width. Data are mean±SD of nine mice in each group. Mice weresacrificed when tumors in the control group reached the maximal sizeallowed by the IACUC Committee at Columbia University.

MRI Imaging and Evaluation of Clinical Response to JNJ-42756493.

Baseline and follow-up imaging assessments were performed on 1.5 TeslaMR imaging systems, including at least axial T1 weighted images beforegadolinium injection, Axial or 3D FLAIR (Fluid-AttenuatedInversion-Recovery), dynamic susceptibility contrast MR perfusion (0.1mmol/kg of gadobutrol), axial and 3D T1 weighted images after gadoliniuminjection. Tumor response was assessed according to the RANO criteria(33). Contrast-enhancing lesion volume was assessed with the help of asemi-automated volumetry tool (SegmentiX), based on shape-detection andthresholding, with control and manual correction of edges whennecessary. Since exclusion of cystic or necrotic portions of the lesionmay be affected by operator subjectivity, both were included forvolumetric and axial measurements.

DSC (dynamic susceptibility contrast) perfusion datasets were processedwith vendor's software suite (Neuroperfusion, Philips), includingcoregistration and rCBV (relative cerebral blood volume) parametric mapsgeneration with 3 different algorithms (Gamma-variate fitting, ArterialInput Function based deconvolution and Model Free).

Results

Detection of FGFR1-TACC1 and FGFR3-TACC3 Fusions in GBM and Grade II-IIIGlioma.

To determine the frequency and molecular features of FGFR-TACC fusionsin human glioma patients, a cohort of 584 GBM and 211 grade II-IIIglioma treated at five Neurooncology centers (Table 12) were screened.108 were grade III (49 IDH wild type, 52 IDH1 mutant and 7 IDH2 mutant)and 103 were grade II (36 IDH wild type, 63 IDH1 mutant and 4 IDH2mutant). The IDH mutational status of 333 GBM was also established andit was determined that 303 harbored wild type IDH1/2 and 30 were mutatedat codon 132 of IDH1. A RT-PCR assay was designed for the detection ofall known and possibly new variants of FGFR1-TACC1 and FGFR3-TACC3fusions that retain the mRNA sequences coding for the key FGFR-TK andTACC domains required for the oncogenic activity of the fusion protein(FIG. 36 and FIGS. 37A-D). Overall, 20 tumors with an FGFR3-TACC3 fusionwere found, of which 17 were GBM (2.9% positives) and 3 lower gradeglioma harboring wild type IDH1/2 genes (3.5% positives). The size ofthe FGFR3-TACC3 RT-PCR amplicons ranged from 928 bp (forFGFR3ex18-TACC3ex13) to 1706 bp (for FGFR3ex18-TACC3ex4). TheFGFR1-TACC1 fusion was detected in one grade II IDH wild type glioma(FIG. 36). Conversely, an IDH1/2 mutant glioma harboring FGFR-TACCfusions (p<0.02) was not found. Sanger sequencing of the fusionamplicons revealed that each FGFR-TACC cDNA joined in-frame the sequencecoding for the entire TK domain upstream of TACC-coding sequences thatinvariably include the coiled-coil TACC domain (FIG. 36). However, anotable variability among FGFR3-TACC3 fusion isoforms was detected,whereby 5 of the identified variants occurred only in individual cases(FIG. 36). Furthermore, 6 fusion transcripts emerged as new variantsthat have not been reported before in human cancer (marked in red inFIG. 36).

Next, suitable PCR primers were designed to map the genomic breakpointcoordinates for 9 FGFR3-TACC3-positive samples for which genomic DNA wasavailable (FIGS. 40 and 41). The genomic breakpoints were successfullyreconstructed by Sanger sequencing and found that they differ for eachof the 9 positive cases. Interestingly, even cases harboring the sameFGFR3-TACC3 transcript splice variants (#4451 and #OPK-14 joining exon17 of FGFR3 to exon 6 of TACC3; #3048 and #4373 joining exon 17 of FGFR3to exon 8 of TACC3; #3808 and #27-1835 joining exon 17 of FGFR3 to exon11 of TACC3) had different genomic breakpoints (FIG. 41). Takentogether, the above findings indicate that the noticeable variabilityamong FGFR3-TACC3 fusion transcripts and genomic breakpoints isefficiently resolved by the RT-PCR screening assay.

Immunostaining Analysis of FGFR3-TACC3-Positive Tumors.

The expression of the FGFR3 fusion protein was analyzed by IHC or IFusing an antibody that recognizes the N-terminal region of FGFR3(FGFR3-N) in 12 GBM and 3 lower grade glioma harboring FGFR3-TACC3fusions for which sufficient tissue was available. Remarkably, each ofthe 15 positive tumors but none of those that had scored negative in theRT-PCR assay, displayed strong positivity for FGFR3 in the vast majorityof tumor cells but not endothelial cells throughout the analyzed tumorsection (FIGS. 37A-H). Notably, IF using an antibody that recognizes anepitope at the C-terminus of TACC3, which is invariably retained withinFGFR3-TACC3 variants (TACC3-C), reproduced the staining pattern of theFGFR3-N antibody in FGFR3-TACC3 positive tumors. Conversely, negative orvery weak staining was obtained in FGFR3-TACC3-positive tumors withantibodies recognizing the regions of FGFR3 (FGFR3 C-terminal region,FGFR3-C) and TACC3 (TACC3 N-terminal region, TACC3-N) constantlyexcluded from FGFR3-TACC3 fusion proteins (FIG. 42A). Consistently,quantitative RT-PCR of GBM harboring FGFR3-TACC3 fusions showed that theexpression of the N-terminal coding region of FGFR3 and the C-terminalcoding region of TACC3 (which are included in the fusion genes) ismarkedly higher than the expression of the C-terminal coding region ofFGFR3 and the N-terminal coding region of TACC3, which are excluded fromthe fusion transcripts (FIG. 42B). One recurrent GBM from a patientwhose tumor had been found positive for FGFR3-TACC3 at the initialdiagnosis and who had recurred after concurrent radiotherapy andtemozolomide treatment was analyzed. The recurrent tumor retained thesame FGFR3-TACC3 fusion gene and protein that was present in theuntreated GBM as determined by RT-PCR-sequencing and FGFR3 IF,respectively (FIG. 43A-C). Although this requires additional evaluation,the retained uniform positivity for FGFR3 in this recurrent GBM suggeststhat targeting the FGFR3-TACC3 fusion protein at relapse is a validtherapeutic strategy.

Clinical and Molecular Characteristics of Glioma Patients withFGFR3-TACC3 Fusions.

Clinical and molecular profiling data were available for 591 patientsincluding 380 GBM (9 with FGFR3-TACC3 fusions) and all 211 lower gradeglioma (3 with FGFR3-TACC3 fusions). Of these 12 patients 5 are malesand 7 females, aged 48y to 82y (median=61y). The molecular profile ofFGFR3-TACC3-positive glioma was determined. To do so, the analysis ofCNVs and somatic mutations of key GBM genes in the dataset was combinedwith the SNP6.0 high-density genomic array analysis of 158 TCGA-derivedGBM samples fully annotated for FGFR3-TACC3 fusion genes (the RNA-seqand/or RT-PCR analysis of these samples had revealed that 5 of themharbor FGFR3-TACC3 fusions) (6). Patients with FGFR3-TACC3 fusionsdisplayed unique characteristics (Table 13). FGFR3-TACC3 fusions weremutually exclusive with EGFR amplification (0/16 vs. 166/411; p=0.0004,FDR q-value corrected for multiple comparisons=0.0012) and showed aclear trend against the presence of the EGFRvIII transcript variant(0/16 vs. 37/219; p=0.083). Conversely, CDK4 amplification wassignificantly more frequent in FGFR3-TACC3-positive tumors (7/16 vs41/408, p=0.0008; FDR q-value=0.0024). A less significant association ofFGFR3-TACC3 fusions was also seen with amplification of MDM2, which asCDK4, maps to chromosome 12q (4/16 vs 24/408, p=0.016; FDRq-value=0.048). No statistical association between FGFR3-TACC3 fusionsand other genetic and epigenetic alterations that commonly occur ingliomas harboring wild type IDH genes was found (CDKN2A deletion, TERTpromoter mutations, gain of chromosome 7p, loss of chromosome 10q andmethylation of the MGMT promoter, Table 13). When compared with the IDHwild type patient population of grade II and grade III glioma and GBM,there was no significant difference in progression free survival (PFS)or overall survival (OS) between patients positive or negative forFGFR3-TACC3 (FIGS. 44A-B). Finally, it was established whether the CNVanalysis of the FGFR3 and TACC3 genomic loci could be used to predictpositivity for FGFR3-TACC3 fusions. The analysis of high-density SNP6.0arrays of the 158 GBM samples from the Atlas-TCGA revealed that 10samples displayed different degrees of copy number gains encompassingthe entire FGFR3 and TACC3 loci (FIG. 45). However, none of themharbored FGFR3-TACC3 fusions. Conversely, the 5 FGFR3-TACC3-positivesamples in the dataset harbor micro-amplification events involving onlythe exons of the FGFR3 gene that are included in the fusion breakpoint.This finding suggests that any CNV survey that is less accurate thanhigh-density SNP arrays, could fail to identify the genomic marksassociated with true FGFR3-TACC3-positive cases.

TABLE 13 Molecular alterations in IDH wild type glioma harboringFGFR3-TACC3 fusions. The table reports the absolute number and frequency(percentage) of individual glioma-specific molecular alterations intumors scoring positive or negative for FGFR3-TACC3 fusions. Theanalysis is done on the Union dataset (TCGA and “Pitié- SalpêtrièreHospital” datasets, see methods for details). Statistically significantassociations are indicated in bold (Fisher Exact test, q-values adjustedwith FDR). N of % of N of % of FGFR3-TACC3 FGFR3-TACC3 FGFR3-TACC3FGFR3-TACC P-value q-value Positive Positive Negative Negative (Fishertest) (FDR) EGFR amplification 0/16 0.0% 166/411 40.4% 4.E−04 0.0012CDK4 amplification 7/16 43.7%  41/408 10.0% 8.E−04 0.0024 MDM2amplification 4/16 25.0%  24/408 5.9% 0.016 0.048 EGFRvIII 0/16 0.0% 37/219 16.9% 0.083 0.25 CDKN2A deletion 4/16 25.0% 188/411 45.7% 0.130.39 Chr. 7p gain 12/15  80.0% 242/374 64.7% 0.28 0.84 Chr. 10q deletion12/16  75.0% 253/420 60.2% 0.3 0.9 TERT promoter 9/11 81.8% 128/16378.5% 0.8 1 mutation MGMT promoter 6/12 50.0%  73/160 45.6% 0.7 1hypermethylation

Preclinical and Clinical Relevance of Targeting FGFR3-TACC3 Fusions.

JNJ-42756493 is a potent, oral pan-FGFR tyrosine kinase inhibitor withIC50 values in the low nanomolar range for all members of the FGFRfamily. It has demonstrated potent antitumor activities in nonclinicalmodels with FGFR aberrations including squamous non-small cell lungcancer, gastric, breast, hepatocellular cancer (HCC), endometrial, andbladder (34, 35). To ask whether JNJ-42756493 is effective in targetingspecifically FGFR-TACC-positive cells, mouse astrocytes expressingFGFR3-TACC3, FGFR3-TACC3 containing a mutation that inactivates thekinase activity of FGFR3 (FGFR3-TACC3-KD), or the empty vector weretreated with JNJ-42756493. The effect of JNJ-42756493 on human gliomastem cells GIC-1123 that harbor the FGFR3-TACC3 gene fusion (6) was alsostudied. These experiments revealed that both mouse astrocytes andGIC-1123 that express FGFR3-TACC3 but not cells expressing the KD mutantfusion or the empty vector are highly sensitive to FGFR inhibition byJNJ-42756493 with an IC50 of 3.03 nM and 1.55 nM, respectively (FIGS.38A-B). Next, the effect of oral treatment with JNJ-42756493 of micebearing xenografts of human GIC-1123 affects tumor growth was tested.Mice were randomized to receive vehicle or JNJ-42756493 (12 mg/kg).Mirroring the in vitro results, JNJ-42756493 elicited a potent growthinhibition of GIC-1123 tumor xenografts (FIGS. 38C-D) with astatistically significant tumor regression after two weeks (p-value ofthe slope calculated from the treatment starting point=0.04). The abovefindings provide a strong foundation for the treatment of GBM patientsharboring FGFR-TACC rearrangements with JNJ-42756493.

Two patients with recurrent GBM harboring FGFR3-TACC3 fusions weretreated with JNJ-42756493 in a first-in-man phase I trial. Patient 1,male aged 52, underwent partial surgical resection of a right parietalGBM, followed by fractionated radiotherapy and concomitant temozolomide(TMZ) as first line treatment (36). The RT-PCR-sequencing analysis ofthe GBM specimen revealed positivity for the FGFR3-TACC3 fusion(FGFR3-exon17-TACC3-exon 6, sample 4451, FIGS. 40 and 41) and theimmunostaining using FGFR3 antibody on paraffin embedded sections showedstrong positivity in a large fraction of tumor cells. After 5 cycles ofTMZ, the patient presented with dizziness and headache and brain MRIrevealed tumor progression (FIG. 39A). At this time the patient wasenrolled in the JNJ-42756493 trial and received JNJ-42756493 (12 mg/dayadministered in cycles of 7 days followed by 7 days off treatment).After 3 weeks the patient reported a marked clinical improvement(complete regression of dizziness and headache). On MRI, the sum ofproduct diameters (RANO criteria, FIG. 39B) and volumetry (FIG. 39C)measured without excluding cystic and necrotic components showed diseasestabilization. However, the tumor mass underwent significant decrease ofthe enhancing parenchyma (−44%) with formation of a cystic portion inthe central core (33). The objective response was further corroboratedby the marked reduction of the extent of tumor vascularity estimated byquantitative analysis of rCBV (relative cerebral blood volume) fromdynamic susceptibility MR perfusion maps (37) (FIG. 39D). Stabilizationlasted for 115 days. During JNJ-42756493 treatment mild and manageabletoxicity was observed (grade I hyperphosphatemia, asthenia, dysgueusia,dry mouth, keratitis, and grade II nail changes). After 4 months, tumorprogressed on MRI locally both on T1 contrast-enhanced area and T2/FLAIRhypersignal. The patient was re-operated and subsequently treated withCCNU. He is still alive, but in progression after 21 months fromdiagnosis and 287 days from the start of the anti-FGFR therapy.

Patient 2 is a 64 years old woman, affected by left parietal GBM,diagnosed by stereotactic biopsy. The tumor was positive for FGFR3-TACC3gene fusion by RT-PCR-sequencing and showed diffuse FGFR3 expression inmost tumor cells (FIGS. 37A, 37C, 37E, sample 4620). The patientreceived as first line treatment fractionated radiotherapy and TMZaccording to the Stupp protocol (36), but after 2 cycles of monthly TMZshe presented with clinical deterioration including progressiveheadaches, right homonymous hemianopsia and memory impairment. Brain MRIperformed 3 and 4 months after the completion of concomitantchemo-radiotherapy revealed tumor progression with increase of the leftparietal mass and the appearance of a small contralateral lesion (FIG.49E). The patient was thus enrolled in the JNJ-42756493 trial (12 mg/dayadministered in cycles of 7 days followed by 7 days off treatment) andshowed clinical improvement after 4 weeks (regression of headaches,visual field defect and memory impairment). Best response was observedafter 104 days of treatment with a 22% reduction of tumor size accordingto the RANO criteria (FIG. 39F) and 28% according to volumetry (FIG.39G). Grade I hyperphosphatemia, nail changes, and mucositis wereobserved. Clinical status remained stable until disease progressionoccurring 134 days after the start of the anti-FGFR. The patient isstill alive and is receiving a third-line chemotherapy with nitrosoureasand bevacizumab.

TABLE 14 Summary of FGFR-TACC fusion transcripts identified in allcancer types. FGFR3-TACC3 fusion variants are ranked according to theirprevalence across any cancer type. The number of FGFR-TACC fusionsidentified in each tumor type, including those identified in the presentstudy, is also indicated. FGFR-TACC Fusion Variants N Cases Tumor TypeFGFR3-TACC3 FGFR3exon17-TACC3exon11 30 Brain Tumors, N = 10 (N = 2,⁸; N= 2,^(9,15); N = 6, Present Study). Bladder Cancer, N = 6 (N =3,^(12,15); N = 3,¹¹). Lung Cancer, N = 13 (N = 4,^(12,15); N = 9,¹⁰).Renal Carcinoma, N = 1,¹⁵. FGFR3exon17-TACC3exon10 18 Brain Tumors, N =5 (N = 1,⁸; N = 1,⁹; N = 3, Present study). Oral Cancer, N = 1,^(12,).Head and Neck Cancer, N = 2,^(12,15). Bladder Cancer, N = 3,⁷. LungCancer, N = 7 (N = 4,⁸; N = 2,¹⁰; N = 1,¹⁴). FGFR3exon17-TACC3exon8 8Brain Tumors, N = 6 (N = 2,⁸. N = 4, Present study). Lung Cancer, N = 2(N = 1,¹⁰; N = 1,¹⁴). FGFR3exon17-TACC3exon4 4 Brain Tumors, N = 2 (N =1,⁹; N = 1,¹⁰). Bladder Cancer, N = 1⁹. Lung Cancer, N = 1¹⁴.FGFR3exon17-TACC3exon6 2 Brain Tumors, N = 2, Present study.FGFR3exon18-TACC3exon4 1 Brain Tumors, N = 1, Present study.FGFR3exon17-TACC3exon9 INS63bp 1 Brain Tumors, N = 1,⁵.FGFR3exon18-TACC3exon9 INS66bp 1 Brain Tumors, N = 1, Present study.FGFR3exon18-TACC3exon5 1 Brain Tumors, N = 1, Present study.FGFR3exon18-TACC3exon5 INS33bp 1 Brain Tumors, N = 1, Present study.INS71bp 1 Lung Cancer, N = 1,¹⁰. FGFR3exon18-TACC3exon13 1 Brain Tumors,N = 1, Present study. FGFR3exon18-TACC3exon11 1 Lung Cancer, N = 1,¹⁰.FGFR1-TACC1 FGFR1exon17-TACC1exon7 5 Brain Tumors, N = 5 (N = 1,⁵; N =3,¹³; N = 1, Present study). FGFR2-TACC2 1 Stomach Adenocarcinoma, N =1¹⁵.

Discussion

FGFR-TACC fusions are potent oncogenic events that when present in braintumor cells confer sensitivity to FGFR inhibitors (6). Since theoriginal identification of recurrent FGFR-TACC fusions in GBM, smallsubgroups of patients harboring FGFR-TACC translocations have beenidentified in several other tumor types (7-15). Here, an unbiasedRT-PCR-sequencing analysis for the identification of all possiblefunctional FGFR-TACC fusion transcripts is reported. The screening of alarge glioma dataset from multiple Institutions not only confirmed thatFGFR-TACC rearrangements occur in ˜3% of human GBM but also revealedthat FGFR-TACC fusions are present in the subgroup of IDH wild typelower grade glioma (grade with prevalence similar to that of GBM. IDHwild type grade II and III glioma have a significantly worse clinicaloutcome than the IDH mutant glioma and manifests molecular and clinicalfeatures that resemble GBM (5). The finding that FGFR-TACC fusions occurin IDH wild type but not IDH mutant glioma provides an important cluefor the molecular characterization of this glioma subtype. Furthermore,the clustering of such potent oncogenic events in IDH wild type gliomaunderscores the particularly aggressive nature of this group of glioma.While it was shown that FGFR-TACC fusions cluster within the poorclinical outcome subgroup of IDH wild type glioma, these translocationsdo not seem to carry prognostic value within the IDH wild type subgroupof glioma patients. Without being bound by theory, the sample size ofpatients harboring FGFR-TACC fusions is too small to draw definitiveconclusions with respect to the impact on survival and larger studiesmay be necessary to clarify the prognostic role of FGFR-TACC fusions inIDH wild type glioma.

Beside mutual exclusivity between IDH1 mutations and FGFR-TACC fusions,the results showed that patients with FGFR3-TACC3 rearrangements lackEGFR amplification and EGFRvIII but are significantly enriched foramplification of CDK4 (and MDM2 to a lesser extent). Knowledge of thesemolecular characteristics will help select those patients who mostlikely harbor FGFR-TACC rearrangements and design combinatorial targetedtherapies that might be more effective in the FGFR-TACC-positive gliomasubgroup.

The molecular screen uncovered 6 new FGFR3-TACC3 fusion events. Togetherwith the previously identified variants, 12 distinct isoforms ofFGFR3-TACC3 have been reported, thus revealing a remarkable variabilityof FGFR3-TACC3 transcripts in human cancer (see Table 14 summarizing thestructure of all the FGFR-TACC variants identified to date). Thestructural heterogeneity of FGFR3-TACC3 fusions is yet more pronouncedat the genomic level, whereby each fusion event harbors distinct genomicbreakpoints, even for identical fusion transcripts. This findingunderscores the notion that targeted genomic analyses are unlikely to besuitable approaches for the molecular diagnosis of FGFR3-TACC3positivity. Conversely, the unbiased identification ofFGFR3-TACC3-positive tumors with the RT-PCR-sequencing assay reportedhere overcomes the limitations of screening only for previouslyidentified FGFR3-TACC3 fusions and provides a simple moleculardiagnostic assay.

Rather than displaying uniform amplifications of the FGFR3 and TACC3genomic loci, FGFR3-TACC3-positive samples harbor small, intragenicmicro-amplification events typically encompassing only the exons of theFGFR3 and TACC3 genes included in the breakpoint (6). This finding isconsistent with the notion that a “fusion breakpoint principle” sustainsthe CNVs of driver gene fusions such as FGFR3-TACC3 in which local CNVstarget exclusively the breakpoint region (38). It is noted that suchsmall and irregular CNVs may easily go undetected from CNV analysesperformed using platforms less sensitive than the high-density SNP6.0genomic arrays. Furthermore, the notion that FGFR3-TACC3-negative GBMmay harbor uniform amplifications across the FGFR3 and TACC3 loci arguesagainst the standard analysis of FGFR3 and/or TACC3 CNVs as a method forthe selection of FGFR3-TACC3-positive tumors.

There is a growing body of evidence supporting the notion that GBM is amarkedly heterogeneous tumor. The formidable degree of intra-tumorheterogeneity of GBM is a potential cause of failure of targetedtherapies in these tumors. In particular, the intra-tumor heterogeneityof GBM has previously been recognized in light of the mosaic expressionof the RTK genes EGFR, PDGFRA and MET by neighboring cells (16-19).Thus, in the majority of GBM, amplification or overexpression ofindividual RTK genes are present in a sub-clonal fraction of tumor cellsand co-exist with amplification/expression of other RTK-coding geneswithin the tumor mass. Therefore, it was essential to determine whethersuch heterogeneity was also present in gliomas harboring FGFR-TACCtranslocations. The immunostaining of FGFR3-TACC3-positive tumorsrevealed that positive specimens manifest strong and uniform expressionof the fusion protein, which is also retained after recurrence. Thisbehavior is reminiscent of other driver chromosome translocations(BCR-ABL, EML4-ALK) and is compatible with the glioma-initiatingfunctions of FGFR-TACC fusions (6). It is also the scenario expected fora driver oncogene whose activity remains essential for tumor maintenanceregardless of secondary genetic alterations that occur during tumorprogression. The strong antitumor effects obtained with JNJ-42756493 inglioma cells harboring FGFR3-TACC3 fusions have built a compellingrationale for the treatment of glioma patients positive for FGFR-TACCrearrangements. JNJ-42756493 is an oral ATP-competitive pan-FGFRselective inhibitor that inhibits tyrosine phosphorylation of activatedFGFR at nanomolar concentrations (34, 35). The enrollment of twopatients with recurrent FGFR3-TACC3-positive GBM in a phase I trial withJNJ-42756493 showed that this treatment has tolerable toxicity and clearanti-tumor activity, thus validating FGFR-TACC as a therapeutic target.Therefore, targeted inhibition of FGFR-TK in preselected IDH wild typeFGFR-TACC-positive glioma may provide clinical benefits for patientswith recurrent glioma who currently lack valuable therapeutic options.In conclusion, described herein is the importance and feasibility ofprospective genotyping for FGFR-TACC fusions in glioma patients andprovided a preliminary evidence of clinical response that warrants theinvestigation of the sensitivity of gliomas harboring FGFR-TACCrearrangements to FGFR kinase inhibition in clinical trials.

REFERENCES

-   1. Medves S, Demoulin J B. Tyrosine kinase gene fusions in cancer:    translating mechanisms into targeted therapies. J Cell Mol Med 2012;    16:237-48.-   2. Mitelman F, Johansson B, Mertens F. The impact of translocations    and gene fusions on cancer causation. Nat Rev Cancer 2007; 7:233-45.-   3. Weathers S P, Gilbert M R. Advances in treating glioblastoma.    F1000Prime Rep. 2014; 6:46.-   4. Omuro A, DeAngelis L M. Glioblastoma and other malignant gliomas:    a clinical review. JAMA 2013; 310:1842-50.-   5. Sanson M, Marie Y, Paris S, Idbaih A, Laffaire J, Ducray F, et    al. Isocitrate dehydrogenase 1 codon 132 mutation is an important    prognostic biomarker in gliomas. J Clin Oncol 2009; 27:4150-4.-   6. Singh D, Chan J M, Zoppoli P, Niola F, Sullivan R, Castano A, et    al. Transforming fusions of FGFR and TACC genes in human    glioblastoma. Science 2012; 337:1231-5.-   7. Cancer Genome Atlas Research N. Comprehensive molecular    characterization of urothelial bladder carcinoma. Nature 2014;    507:315-22.-   8. Majewski I J, Mittempergher L, Davidson N M, Bosma A, Willems S    M, Horlings H M, et al. Identification of recurrent FGFR3 fusion    genes in lung cancer through kinome-centred RNA sequencing. J Pathol    2013; 230:270-6.-   9. Parker B C, Annala M J, Cogdell D E, Granberg K J, Sun Y, Ji P,    et al. The tumorigenic FGFR3-TACC3 gene fusion escapes miR-99a    regulation in glioblastoma. J Clin Invest 2013; 123:855-65.-   10. Wang R, Wang L, Li Y, Hu H, Shen L, Shen X, et al. FGFR1/3    Tyrosine Kinase Fusions Define a Unique Molecular Subtype of    Non-Small Cell Lung Cancer. Clin Cancer Res 2014; 20:4107-14.-   11. Williams S V, Hurst C D, Knowles M A. Oncogenic FGFR3 gene    fusions in bladder cancer. Hum Mol Genet 2013; 22:795-803.-   12. Wu Y M, Su F, Kalyana-Sundaram S, Khazanov N, Ateeq B, Cao X, et    al. Identification of targetable FGFR gene fusions in diverse    cancers. Cancer Discov 2013; 3:636-47.-   13. Zhang J, Wu G, Miller C P, Tatevossian R G, Dalton J D, Tang B,    et al. Whole-genome sequencing identifies genetic alterations in    pediatric low-grade gliomas. Nat Genet 2013; 45:602-12.-   14. Capelletti M, Dodge M E, Ercan D, Hammerman P S, Park S I, Kim    J, et al. Identification of Recurrent FGFR3-TACC3 Fusion Oncogenes    from Lung Adenocarcinoma. Clin Cancer Res 2014, DOI:    10.1158/1078-0432. CCR-14-1337; in press.-   15. Stransky N, Cerami E, Schalm S, Kim J L, Lengauer C. The    landscape of kinase fusions in cancer. Nat Commun 2014; 5:4846.-   16. Inda M M, Bonavia R, Mukasa A, Narita Y, Sah D W, Vandenberg S,    et al. Tumor heterogeneity is an active process maintained by a    mutant EGFR-induced cytokine circuit in glioblastoma. Genes Dev    2010; 24:1731-45.-   17. Snuderl M, Fazlollahi L, Le L P, Nitta M, Zhelyazkova B H,    Davidson C J, et al. Mosaic amplification of multiple receptor    tyrosine kinase genes in glioblastoma. Cancer Cell 2011; 20:810-7.-   18. Ene C I, Fine H A. Many tumors in one: a daunting therapeutic    prospect. Cancer Cell 2011; 20:695-7.-   19. Sottoriva A, Spiteri I, Piccirillo S G, Touloumis A, Collins V    P, Marioni J C, et al. Intratumor heterogeneity in human    glioblastoma reflects cancer evolutionary dynamics. Proc Natl Acad    Sci USA 2013; 110:4009-14.-   20. Kindich R, Florl A R, Jung V, Engers R, Muller M, Schulz W A, et    al.

Application of a modified real-time PCR technique for relative gene copynumber quantification to the determination of the relationship betweenNKX3.1 loss and MYC gain in prostate cancer. Clin Chem 2005; 51:649-52.

-   21. Bulusu K C, Tym J E, Coker E A, Schierz A C, Al-Lazikani B.    canSAR: updated cancer research and drug discovery knowledgebase.    Nucleic Acids Res 2014; 42:D1040-7.-   22. Reyes-Botero G, Giry M, Mokhtari K, Labussiere M, Idbaih A,    Delattre J Y, et al. Molecular analysis of diffuse intrinsic    brainstem gliomas in adults. J Neurooncol 2014; 116:405-11.-   23. Labussière M B B, Mokhtari K, Di Stefano A L, Rahimian A,    Rossetto M, Ciccarino P, Saulnier O, Paterra R, Marie Y, Finocchiaro    G, Sanson M. Combined analysis of TERT, EGFR and IDH status define    distinct prognostic glioblastoma classes. Neurology 2014; 83:1200-6.-   24. Quillien V, Lavenu A, Karayan-Tapon L, Carpentier C, Labussiere    M, Lesimple T, et al. Comparative assessment of 5 methods    (methylation-specific polymerase chain reaction, MethyLight,    pyrosequencing, methylation-sensitive high-resolution melting, and    immunohistochemistry) to analyze    O6-methylguanine-DNA-methyltranferase in a series of 100    glioblastoma patients. Cancer 2012; 118:4201-11.-   25. Idbaih A, Aimard J, Boisselier B, Marie Y, Paris S, Criniere E,    et al. Epidermal growth factor receptor extracellular domain    mutations in primary glioblastoma. Neuropathol Appl Neurobiol 2009;    35:208-13.-   26. Idbaih A, Marie Y, Lucchesi C, Pierron G, Manie E, Raynal V, et    al. BAC array CGH distinguishes mutually exclusive alterations that    define clinicogenetic subtypes of gliomas. Int J Cancer 2008;    122:1778-86.-   27. Gonzalez-Aguilar A, Idbaih A, Boisselier B, Habbita N, Rossetto    M, Laurenge A, et al. Recurrent mutations of MYD88 and TBL1XR1 in    primary central nervous system lymphomas. Clin Cancer Res 2012;    18:5203-11.-   28. Olshen A B, Venkatraman E S, Lucito R, Wigler M. Circular binary    segmentation for the analysis of array-based DNA copy number data.    Biostatistics 2004; 5:557-72.-   29. Hoang-Xuan K, He J, Huguet S, Mokhtari K, Marie Y, Kujas M, et    al. Molecular heterogeneity of oligodendrogliomas suggests    alternative pathways in tumor progression. Neurology 2001;    57:1278-81.-   30. Houillier C, Lejeune J, Benouaich-Amiel A, Laigle-Donadey F,    Criniere E, Mokhtari K, et al. Prognostic impact of molecular    markers in a series of 220 primary glioblastomas. Cancer 2006;    106:2218-23.-   31. Goldman M, Craft B, Swatloski T, Ellrott K, Cline M, Diekhans M,    et al. The UCSC Cancer Genomics Browser: update 2013. Nucleic Acids    Res 2013; 41:D949-54.-   32. Brennan C W, Verhaak R G, McKenna A, Campos B, Noushmehr H,    Salama S R, et al. The somatic genomic landscape of glioblastoma.    Cell 2013; 155:462-77.-   33. Wen P Y, Macdonald D R, Reardon D A, Cloughesy T F, Sorensen A    G, Galanis E, et al. Updated response assessment criteria for    high-grade gliomas: response assessment in neuro-oncology working    group. J Clin Oncol 2010; 28:1963-72.-   34. Bahleda R, Dienstmann R, Adamo B, Gazzah A, Infante J R, Zhong    B, et al. Phase 1 study of JNJ-42756493, a pan-fibroblast growth    factor receptor (FGFR) inhibitor, in patients with advanced solid    tumors. J Clin Oncol 2014; 32:suppl; abstr 2501.-   35. Squires M, Ward G, Saxty G, Berdini V, Cleasby A, King P, et al.    Potent, selective inhibitors of fibroblast growth factor receptor    define fibroblast growth factor dependence in preclinical cancer    models. Mol Cancer Ther 2011; 10:1542-52.-   36. Stupp R, Mason W P, van den Bent M J, Weller M, Fisher B,    Taphoorn M J, et al. Radiotherapy plus concomitant and adjuvant    temozolomide for glioblastoma. N Engl J Med 2005; 352:987-96.-   37. Law M, Yang S, Babb J S, Knopp E A, Golfinos J G, Zagzag D, et    al. Comparison of cerebral blood volume and vascular permeability    from dynamic susceptibility contrast-enhanced perfusion M R imaging    with glioma grade. AJNR Am J Neuroradiol 2004; 25:746-55.-   38. Wang X S, Prensner J R, Chen G, Cao Q, Han B, Dhanasekaran S M,    et al. An integrative approach to reveal driver gene fusions from    paired-end sequencing data in cancer. Nat Biotechnol 2009;    27:1005-11.

What is claimed is:
 1. An antibody or antigen-binding fragment thereof,that specifically binds to a purified fusion protein comprising atyrosine kinase domain of an FGFR protein fused to the TACC domain of atransforming acidic coiled-coil-containing (TACC) protein.
 2. Theantibody or antigen-binding fragment of claim 1, wherein the FGFRprotein is FGFR1, FGFR2, FGFR3, or FGFR4.
 3. The antibody orantigen-binding fragment of claim 1, wherein the TACC protein is TACC1,TACC2, or TACC3.
 4. The antibody or antigen-binding fragment of claim 1,wherein the fusion protein is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3.5. The antibody or antigen-binding fragment of claim 4, wherein theFGFR1-TACC1 fusion protein comprises the amino acid sequence of SEQ IDNO:
 150. 6. The antibody or antigen-binding fragment of claim 4, whereinthe FGFR3-TACC3 fusion protein comprises the amino acid sequence of SEQID NO: 79, 158, 159, 160, or
 161. 7. A composition for decreasing in asubject the expression level or activity of a fusion protein comprisingthe tyrosine kinase domain of an FGFR protein fused to the TACC domainof a TACC protein, the composition in an admixture of a pharmaceuticallyacceptable carrier comprising an inhibitor of the fusion protein.
 8. Thecomposition of claim 7, wherein the TACC protein is TACC1, TACC2, orTACC3.
 9. The composition of claim 7, wherein the inhibitor comprises anantibody that specifically binds to a FGFR-TACC fusion protein or afragment thereof; a small molecule that specifically binds to a FGFRprotein; a small molecule that specifically binds to a TACC protein; anantisense RNA or antisense DNA that decreases expression of a FGFR-TACCfusion polypeptide; a siRNA that specifically targets a FGFR-TACC fusiongene; or a combination thereof.
 10. The composition of claim 7, whereinthe FGFR protein is FGFR1, FGFR2, FGFR3, or FGFR4.
 11. The compositionof claim 7, wherein the FGFR-TACC fusion protein is FGFR1-TACC1,FGFR2-TACC2, or FGFR3-TACC3.
 12. The composition of claim 9, wherein thesmall molecule that specifically binds to a FGFR protein comprisesAZD4547, NVP-BGJ398, PD173074, NF449, TK1258, BIBF-1120, BMS-582664,AZD-2171, TSU68, AB1010, AP24534, E-7080, LY2874455, or a combinationthereof.
 13. A method for decreasing in a subject in need thereof theexpression level or activity of a fusion protein comprising the tyrosinekinase domain of an FGFR protein fused to the TACC domain of a TACCprotein, the method comprising: (a) administering to the subject atherapeutic amount of a composition of claim 7; and (b) determiningwhether the fusion protein expression level or activity is decreasedcompared to fusion protein expression level or activity prior toadministration of the composition, thereby decreasing the expressionlevel or activity of the fusion protein.
 14. A method of decreasinggrowth of a solid tumor in a subject in need thereof, the methodcomprising administering to the subject an effective amount of a FGFRfusion molecule inhibitor, wherein the inhibitor decreases the size ofthe solid tumor and, wherein the FGFR fusion comprises the tyrosinekinase domain of FGFR fused to the TACC domain of TACC.
 15. The methodof claim 14, wherein the solid tumor comprises glioblastoma multiforme,breast cancer, lung cancer, prostate cancer, or colorectal carcinoma.16. The method of claim 14, wherein the inhibitor comprises an antibodythat specifically binds to a FGFR-TACC fusion protein or a fragmentthereof; a small molecule that specifically binds to a FGFR protein; asmall molecule that specifically binds to a TACC protein; an antisenseRNA or antisense DNA that decreases expression of a FGFR-TACC fusionpolypeptide; a siRNA that specifically targets a FGFR-TACC fusion gene;or a combination thereof.
 17. The method of claim 13 or 14, wherein theFGFR is FGFR1, FGFR2, FGFR3, or FGFR4.
 18. The method of claim 13 or 16,wherein the fusion protein is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3.19. The method of claim 16, wherein the small molecule that specificallybinds to a FGFR protein comprises AZD4547, NVP-BGJ398, PD173074, NF449,TK1258, BIBF-1120, BMS-582664, AZD-2171, TSU68, AB1010, AP24534, E-7080,LY2874455, or a combination thereof.
 20. A diagnostic kit fordetermining whether a sample from a subject exhibits a presence of aFGFR fusion, the kit comprising at least one oligonucleotide thatspecifically hybridizes to a FGFR fusion, or a portion thereof, andwherein the FGFR fusion comprises the tyrosine kinase domain of FGFRfused to the TACC domain of TACC.
 21. The kit of claim 20, wherein theoligonucleotides comprise a set of nucleic acid primers or in situhybridization probes.
 22. The kit of claim 20, wherein theoligonucleotide comprises SEQ ID NO: 162, 163, 164, 165, 166, 167, 168,169, or a combination thereof.
 23. The kit of claim 21, wherein theprimers prime a polymerase reaction only when a FGFR fusion is present.24. The kit of claim 20, wherein the determining comprises genesequencing, selective hybridization, selective amplification, geneexpression analysis, or a combination thereof.
 25. A diagnostic kit fordetermining whether a sample from a subject exhibits a presence of aFGFR fusion protein, the kit comprising an antibody that specificallybinds to a FGFR fusion protein comprising SEQ ID NO: 79, 85, 86, 87, 88,89, 150, 158, 159, 160, or 161, wherein the antibody will recognize theprotein only when a FGFR fusion protein is present, and wherein the FGFRfusion protein comprises a tyrosine kinase domain of an FGFR proteinfused to the TACC domain of a transforming acidic coiled-coil-containing(TACC) protein.
 26. The kit of claim 25, wherein the antibody isdirected to an FGFR fusion protein comprising SEQ ID NO: 79, 85, 86, 87,88, 89, 150, 158, 159, 160, or
 161. 27. The kit of claim 20 or 25,wherein the FGFR is FGFR1, FGFR2, FGFR3, or FGFR4.
 28. The kit of claim20 or 25, wherein the FGFR fusion is FGFR1-TACC1, FGFR2-TACC2, orFGFR3-TACC3.
 29. A method for detecting the presence of a FGFR fusion ina human subject, wherein the FGFR fusion comprises the tyrosine kinasedomain of FGFR fused to the TACC domain of TACC, the method comprising:(a) obtaining a biological sample from the human subject; and (b)detecting whether or not there is a FGFR fusion present in the subject.30. The method of claim 29, wherein the detecting comprises measuringFGFR fusion protein levels by ELISA using an antibody directed to SEQ IDNO: 79, 85, 86, 87, 88, 89, 150, 158, 159, 160, or 161; western blotusing an antibody directed to SEQ ID NO: 79, 85, 86, 87, 88, 89, 150,158, 159, 160, or 161; mass spectroscopy, isoelectric focusing, or acombination thereof.
 31. The method of claim 29, wherein the detectingof step (b) comprises detecting whether or not there is a nucleic acidsequence encoding a FGFR fusion protein in the subject.
 32. The methodof claim 31, wherein the nucleic acid sequence comprises any one of SEQID NOS: 1-77, 80-84, or 95-145.
 33. The method of claim 31, wherein thedetecting comprises using hybridization, amplification, or sequencingtechniques to detect a FGFR fusion.
 34. The method of claim 33, whereinthe amplification uses primers comprising SEQ ID NO: 162, 163, 164, 165,166, 167, 168, or
 169. 35. The method of claim 29 or 31, wherein theFGFR is FGFR1, FGFR2, FGFR3, or FGFR4.
 36. The method of claim 29 or 31,wherein the FGFR fusion is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3. 37.A method of identifying a compound that decreases the oncogenic activityof a FGFR-TACC fusion, the method comprising: a) transducing a cellcultured in vitro with FGFR-TACC DNA; b) contacting a cell with a ligandsource for an effective period of time; and c) determining whether thecells acquire the ability to grow in anchorage-independent conditions,form multi-layered foci, or a combination thereof, compared to cellscultured in the absence of the test compound.
 38. A purified fusionprotein comprising the tyrosine kinase domain of an FGFR protein fusedto the TACC domain of a transforming acidic coiled-coil-containing(TACC) protein.
 39. The purified fusion protein of claim 38, wherein theFGFR protein is FGFR1, FGFR2, FGFR3, or FGFR4.
 40. The purified fusionprotein of claim 38, wherein the TACC protein is TACC1, TACC2, or TACC3.41. The purified fusion protein of claim 38, wherein the fusion proteinis FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3.
 42. The purified fusionprotein of claim 38, wherein the fusion protein comprises SEQ ID NO: 79,SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, or SEQ ID NO:
 161. 43.The purified fusion protein of claim 38, wherein the fusion protein hasa breakpoint comprising at least 3 consecutive amino acids from aminoacids 730-758 of SEQ ID NO: 90 and comprising at least 3 consecutiveamino acids from amino acids 549-838 of SEQ ID NO:
 92. 44. The purifiedfusion protein of claim 38, wherein the fusion protein has a breakpointcomprising SEQ ID NO: 78, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87,or SEQ ID NO:89.
 45. The purified fusion protein of claim 38, whereinthe fusion protein comprises SEQ ID NO:
 150. 46. The purified fusionprotein of claim 38, wherein the fusion protein has a breakpointcomprising at least 3 consecutive amino acids from amino acids 746-762of SEQ ID NO: 146 and comprising at least 3 consecutive amino acids fromamino acids 572-590 of SEQ ID NO:
 148. 47. The purified fusion proteinof claim 38, wherein the fusion protein has a breakpoint comprising SEQID NO:
 88. 48. A cDNA encoding a fusion protein comprising the tyrosinekinase domain of FGFR fused to the TACC domain of TACC.
 49. The cDNA ofclaim 48, wherein the FGFR is FGFR1, FGFR2, FGFR3, or FGFR4.
 50. ThecDNA of claim 48, wherein the TACC is TACC1, TACC2, or TACC3.
 51. ThecDNA of claim 48, wherein the fusion protein is FGFR1-TACC1,FGFR2-TACC2, or FGFR3-TACC3.
 52. The cDNA of claim 48, wherein the cDNAcomprises SEQ ID NO:
 94. 53. The cDNA of claim 48, where in the cDNA hasa breakpoint comprising at least 9 consecutive in-frame nucleotides fromnucleotides 2443-2530 of SEQ ID NO: 91 and comprising at least 9consecutive in-frame nucleotides from nucleotides 1800-2847 of SEQ IDNO:
 93. 54. The cDNA of claim 48, where in the cDNA has a breakpointcomprising any one of SEQ ID NOs: 1-77.
 55. The cDNA of claim 48,wherein the cDNA comprises SEQ ID NO:
 151. 56. The cDNA of claim 48,where in the cDNA has a breakpoint comprising at least 9 consecutivein-frame nucleotides from nucleotides 3178-3228 of SEQ ID NO: 147 andcomprising at least 9 consecutive in-frame nucleotides from nucleotides2092-2794 of SEQ ID NO:
 149. 57. The cDNA of claim 48, where in the cDNAhas a breakpoint comprising SEQ ID NO:
 83. 58. The cDNA of claim 48,comprising a combination of exons 1-16 of FGFR3 spliced 5′ to acombination of exons 8-16 of TACC3, wherein a breakpoint occurs in: a)any one of exons 1-16 of FGFR3 and any one of exons 8-16 of TACC3; b)any one of introns 1-16 of FGFR3 and any one of exons 8-16 of TACC3; c)any one of exons 1-16 of FGFR3 and any one of introns 7-16 of TACC3; ord) any one of introns 1-16 of FGFR3 and any one of introns 7-16 ofTACC3.
 59. The cDNA of claim 48, comprising a combination of exons 1-17of FGFR1 spliced 5′ to a combination of exons 7-13 of TACC1, wherein abreakpoint occurs in any one of exons 1-17 of FGFR3 and any one of exons7-13 of TACC3.
 60. The cDNA of claim 48, comprising a combination ofexons 1-18 of FGFR2 spliced 5′ to a combination of exons 1-23 of TACC2.